Methods for using compositions comprising heat shock proteins or alpha-2-macroglobulin in the treatment of cancer and infectious disease

ABSTRACT

The present invention relates to methods and compositions for the prevention and treatment of infectious diseases, and cancers. The methods of the invention comprises administering (a) a composition comprising a population of complexes of antigenic proteins or antigenic peptides derived from antigenic cells or viral particles and one or more different heat shock proteins; and (b) a non-heat shock protein and non-alpha-2-macroglobulin-based treatment modality. The population or the protein preparation used to produce the antigenic peptides comprises at least 50% of the different proteins or at least 50 different proteins of the antigenic cells or viral particles. Methods for making antigenic peptides comprise digesting a protein preparation of antigenic cells, a cellular fraction thereof, or of viral particles with one or more proteases, or exposing the protein preparation to ATP, guanidium hydrochloride, and/or acidic conditions.

[0001] The present application claims the benefit of U.S. provisionalapplication No. 60/449,001, filed Feb. 20, 2003, which is incorporatedby reference herein in its entirety.

[0002] This invention was made with government support under grantnumber CA/A184479 awarded by the National Institutes of Health. TheUnited States Government has certain rights in the invention.

1. INTRODUCTION

[0003] The present invention relates to methods and compositions for theprevention and treatment of infectious diseases, and primary andmetastatic neoplastic diseases. In the practice of the prevention andtreatment of infectious diseases and cancer, compositions comprisingcytosolic and membrane-derived proteins from antigenic cells and/or thedigestion products thereof, are complexed to heat shock proteins and/oralpha-2-macroglobulin to augment the immune response to tumors andinfectious agents. The uses of such compositions in combination withother treatment modalities are also encompassed.

2. BACKGROUND OF THE INVENTION 2.1. Heat Shock Proteins

[0004] Heat shock proteins (HSPs), also referred to as stress proteins,were first identified as proteins synthesized by cells in response toheat shock. HSPs have been classified into five families, based onmolecular weight, HSP100 , HSP90, HSP70, HSP60, and smHSP. Many membersof these families were found subsequently to be induced in response toother stressful stimuli including nutrient deprivation, metabolicdisruption, oxygen radicals, and infection with intracellular pathogens(see Welch, May 1993, Scientific American 56-64; Young, 1990, Annu. Rev.Immunol. 8:401-420; Craig, 1993, Science 260:1902-1903; Gething et al.,1992, Nature 355:33-45; and Lindquist et al., 1988, Annu. Rev. Genetics22:631-677).

[0005] Studies on the cellular response to heat shock and otherphysiological stresses revealed that the HSPs are involved not only incellular protection against these adverse conditions, but also inessential biochemical and immunological processes in unstressed cells.HSPs accomplish different kinds of chaperoning functions. For example,members of the HSP70 family, located in the cell cytoplasm, nucleus,mitochondria, or endoplasmic reticulum (Lindquist et al., 1988, Ann.Rev. Genetics 22:631-677), are involved in the presentation of antigensto the cells of the immune system, and are also involved in thetransfer, folding and assembly of proteins in normal cells. HSPs arecapable of binding proteins or peptides, and releasing the boundproteins or peptides in the presence of adenosine triphosphate (ATP) oracidic conditions (Udono and Srivastava, 1993, J. Exp. Med.178:1391-1396).

[0006] Srivastava et al. demonstrated immune response tomethylcholanthrene-induced sarcomas of inbred mice (1988, Immunol. Today9:78-83). In these studies, it was found that the molecules responsiblefor the individually distinct immunogenicity of these tumors wereglycoproteins of 96 kDa (gp96) and intracellular proteins of 84 to 86kDa (Srivastava et al., 1986, Proc. Natl. Acad. Sci. USA 83:3407-3411;Ullrich et al., 1986, Proc. Natl. Acad. Sci. USA 83:3121-3125).Immunization of mice with gp96 or p84/86 isolated from a particulartumor rendered the mice immune to that particular tumor, but not toantigenically distinct tumors. Isolation and characterization of genesencoding gp96 and p84/86 revealed significant homology between them, andshowed that gp96 and p84/86 were, respectively, the endoplasmicreticular and cytosolic counterparts of the same heat shock proteins(Srivastava et al., 1988, Immunogenetics 28:205-207; Srivastava et al.,1991, Curr. Top. Microbiol. Immunol. 167:109-123). Further, HSP70 wasshown to elicit immunity to the tumor from which it was isolated but notto antigenically distinct tumors. However, HSP70 depleted of peptideswas found to lose its immunogenic activity (Udono and Srivastava, 1993,J. Exp. Med. 178:1391-1396). These observations suggested that the heatshock proteins are not immunogenic per se, but form noncovalentcomplexes with antigenic peptides, and the complexes can elicit specificimmunity to the antigenic peptides (Srivastava, 1993, Adv. Cancer Res.62:153-177; Udono et al., 1994, J. Immunol., 152:5398-5403; Suto et al.,1995, Science 269:1585-1588).

[0007] Noncovalent complexes of HSPs and peptide, purified from cancercells, can be used for the treatment and prevention of cancer and havebeen described in PCT publications WO 96/10411, dated Apr. 11, 1996, andWO 97/10001, dated Mar. 20, 1997 (U.S. Pat. No. 5,750,119 issued May 12,1998, and U.S. Pat. No. 5,837,251 issued Nov. 17, 1998, respectively,each of which is incorporated by reference herein in its entirety). Theisolation and purification of HSP-peptide complexes has been described,for example, from pathogen-infected cells, and used for the treatmentand prevention of infection caused by the pathogen, such as viruses, andother intracellular pathogens, including bacteria, protozoa, fungi andparasites (see, for example, PCT Publication WO 95/24923, dated Sep. 21,1995). Immunogenic stress protein-antigen complexes can also be preparedby in vitro complexing of stress protein and antigenic peptides, and theuses of such complexes for the treatment and prevention of cancer andinfectious diseases has been described in PCT publication WO 97/10000,dated Mar. 20, 1997 (U.S. Pat. No. 6,030,618 issued Feb. 29, 2000). Theuse of stress protein-antigen complexes for sensitizing antigenpresenting cells in vitro for use in adoptive immunotherapy is describedin PCT publication WO 97/10002, dated Mar. 20, 1997 (see also U.S. Pat.No. 5,985,270 issued Nov. 16, 1999).

2.2. Alpha-2-Macroglobulin

[0008] The α-macroglobulins are members of a protein superfamily ofstructurally related proteins which also comprises complement componentsC3, C4 and C5. The human plasma protein alpha-2-macroglobulin (α2M) is a720 kDa homotetrameric protein primarily known as proteinase inhibitorand plasma and inflammatory fluid proteinase scavenger molecule (forreview see Chu and Pizzo, 1994, Lab. Invest. 71:792). α2M is synthesizedas a precursor having 1474 amino acid residues. The first 23 amino acidsfunction as a signal sequence that is cleaved to yield a mature proteinwith 1451 amino acid residues (Kan et al., 1985, Proc. Natl. Acad. Sci.U.S.A. 82:2282-2286).

[0009] α2M promiscuously binds to proteins and peptides withnucleophilic amino acid side chains in a covalent manner (Chu et al.,1994, Ann. N.Y. Acad. Sci. 737:291-307) and targets them to cells whichexpress a α2M receptor (α2MR) (Chu and Pizzo, 1993, J. Immunol. 150:48).Binding of α2M to the α2M receptor is mediated by the carboxy-terminalportion of α2M (Holtet et al., 1994, FEBS Lett. 344:242-246) and keyresidues have been identified (Nielsen et al., 1996, J. Biol. Chem.271:12909-12912).

[0010] Generally known for inhibiting protease activity, α2M binds to avariety of proteases through multiple binding sites (see, e.g., Hall etal., 1981, Biochem. Biophys. Res. Commun. 100(1):8-16). Proteaseinteraction with α2M results in a complex structural rearrangementcalled transformation, which is the result of a cleavage within the“bait” region of α2M after the proteinase becomes “trapped” bythioesters. The conformational change exposes residues required forreceptor binding, allowing the α2M-proteinase complex to bind to theα2MR. Methylamine can induce similar conformational changes and cleavageas that induced by proteinases. The uncleaved form of α2M, which is notrecognized by the receptor, is often referred to as the “slow” form(s-α2M). The cleaved form is referred to as the “fast” form (f-α2M)(reviewed by Chu et al., 1994, Ann. N.Y. Acad. Sci. 737:291-307).Recently, it has also been shown that the α2MR can bind to HSPs, such asgp96, hsp90, hsp70, and calreticulin (Basu et al., 2001, Immunity14(3):303-13).

[0011] Studies have shown that in addition to its proteinase-inhibitoryfunctions, α2M, when complexed to antigens, can enhance the antigens'ability to be taken up by antigen presenting cells such as macrophagesand presented to T cell hybridomas in vitro by up to two orders ofmagnitude (Chu and Pizzo, 1994, Lab. Invest. 71:792), and to induce Tcell proliferation (Osada et al., 1987, Biochem. Biophys. Res.Commun.146:26-31). Further evidence suggests that complexing antigenwith α2M enhances antibody production by crude spleen cells in vitro(Osada et al., 1988, Biochem. Biophys. Res. Commun. 150:883), elicits anin vivo antibody responses in experimental rabbits (Chu et al., 1994, J.Immunol. 152:1538-1545) and mice (Mitsuda et al., 1993, Biochem.Biophys. Res. Commun. 101: 1326-1331). α2M-antigenic peptide complexeshave also been shown to induce a cytotoxic T cell response in vivo(Binder et al., 2001, J. Immunol. 166:4698-49720).

3. SUMMARY OF THE INVENTION

[0012] The present invention encompasses the making and using ofcomplexes of antigenic proteins and peptides and heat shock protein(HSP) or alpha-2-macroglobulin (α2M) for the prevention and treatment ofcancer and infectious disease. Preferably, the complexes are used incombination with at least one non-heat shock protein andnon-alpha-2-macroglobulin-based treatment modality.

[0013] In one embodiment, the invention uses complexes of HSPs and apopulation of antigenic proteins of antigenic cells or viral particlesprepared by a method that involves complexing a population of antigenicproteins derived from antigenic cells or viral particles to one or moredifferent heat shock proteins in vitro, wherein the population comprisesat least 50% of the different proteins or at least 50 different proteinsthat are present in the antigenic cells or viral particles, or presentin a cellular fraction of the antigenic cells.

[0014] In another embodiment, the complexes are prepared by a methodthat comprises contacting the protein preparation in vitro with one ormore different heat shock proteins under conditions such that proteinsin the protein preparation are complexed to the heat shock proteins.

[0015] In yet another embodiment, the invention provides uses ofcomplexes comprising HSPs and a population of antigenic peptides ofantigenic cells or viral particles, wherein the population of antigenicpeptides is generated by a method comprising digesting a proteinpreparation of antigenic cells, a cellular fraction thereof, or viralparticles with either a protease or a plurality of different proteasesseparately. The population of antigenic peptides can also be generatedby a method comprising exposing a protein preparation of antigeniccells, a cellular fraction thereof, or viral particles to ATP, guanidiumhydrochloride, and/or acidic conditions sufficient to elute antigenicpeptides from protein complexes present in the protein preparation. Theantigenic peptides generated by either or both methods are complexed toone or more different HSPs in vitro.

[0016] In yet another embodiment, the invention provides uses ofcomplexes of α2M and a population of antigenic proteins of antigeniccells. The complexes are prepared by a method that involves complexing apopulation of antigenic proteins derived from antigenic cells or viralparticles to α2M in vitro, wherein the population comprises at least 50%of the different proteins or at least 50 different proteins that arepresent in the antigenic cells or viral particles, or present in acellular fraction of the antigenic cells. In another embodiment, themethod comprises contacting the protein preparation in vitro with α2Munder conditions such that proteins in the protein preparation arecomplexed to α2M.

[0017] In yet another embodiment, the invention provides uses ofcomplexes comprising α2M and a population of antigenic peptides ofantigenic cells or viral particles, wherein the population of antigenicpeptides is generated by a method comprising digesting a proteinpreparation of antigenic cells, a cellular fraction thereof, or viralparticles, with either a protease or a plurality of different proteasesseparately. The population of antigenic peptides can also be generatedby a method comprising exposing a protein preparation of antigeniccells, a cellular fraction thereof, or viral particles, with ATP,guanidium hydrochloride, and/or acidic conditions. The antigenicpeptides generated by either or both methods are complexed to α2M invitro.

[0018] In various embodiments, the antigenic cells can be cancer cells,or cells infected with a pathogen or infectious agent, and preferablyhuman cells. The antigenic cells can also be cells of a pathogen orinfectious agent, or variants thereof. The antigenic proteins/peptidescan be prepared from cancer cells or cells infected with a pathogen thatare antigenically related to the cancer or infectious diseases. Apathogen or infectious agent, including viral particles can also be usedto prepare the antigenic peptides. The protein preparation of theantigenic cells may comprise only cytosolic proteins, onlymembrane-derived proteins, or both cytosolic and membrane-derivedproteins. The protein preparation may be a crude, unfractionated celllysate. In a specific embodiment, the protein preparation can be made bylysing the antigenic cells, removing cell debris and non-proteinaceousmaterials, and optionally purifying the proteins, by methods known inthe art. In certain embodiments, the protein preparation has not beensubjected to any method of preparation that selectively removes orretains one or more particular protein from the other proteins in theantigenic cells.

[0019] In certain embodiments, the protein preparation of the antigeniccells, a cellular fraction thereof, or viral particles can be digestedby a variety of proteases, such as but not limited to trypsin,Staphylococcal peptidase I (also known as protease V8), chymotrypsin,pepsin, cathepsin G, thermolysin, elastase, and papain, under conditionssuitable for enzymatic reaction. The extent of the digestion can bemonitored by taking a sample and analyzing it by known techniques fordetermining the length of peptides. It is preferable that the digestingstep is carried out under conditions such that the resulting populationof peptides which comprises antigenic peptides, have an average size offrom about 7 amino acid residues to about 20 amino acid residues. It isalso desirable to generate from a protein preparation differentpopulations of peptides by digesting aliquots of the protein preparationwith different proteases. The peptides resulting from the differentdigests may be combined before complexing to HSP or α2M. Beforecomplexing the population of peptides which comprises antigenic peptidesto HSP or α2M, it may be desirable to inactivate or separate theprotease from the population of peptides, and optionally purify thepopulation of peptides.

[0020] In certain embodiments, the protein preparation of the antigeniccells, a cellular fraction thereof, or viral particles are contactedwith adenosine triphosphate (ATP), guanidium hydrochloride, and/oracidic conditions such that antigenic peptides can be eluted without theneed to isolate HSP complexes or α2M complexes initially. The antigenicpeptides eluted by this method comprise peptides that are endogenouslyassociated with HSPs, α2M, and MHC class I and II molecules.

[0021] In various embodiments of the invention, depending on the methodused to complex the population of antigenic peptides to HSP or α2M invitro, the reaction can result in the antigenic peptides complexed toHSP or α2M by either a covalent bond or non-covalent bond. Heat shockproteins that are contemplated for complexing include but not limited toHSP 60, HSP70, HSP 90, gp96, calreticulin, grp78 (or BiP), proteindisulfide isomerase (PDI), HSP 110, and grp 170. Human HSPs and humanα2M are generally preferred. The complexes of HSP or α2M and antigenicpeptides formed in vitro can be further purified before their use in oras a therapeutic or prophylactic composition. Such compositions mayfurther comprises an adjuvant. Kits for combination therapy comprisingHSP and/or α2M, antigenic cells, protein preparations, and/or proteases,and additional treatment modalities are also provided.

[0022] In another aspect, a method is provided for treating orpreventing a type of cancer or infectious disease, comprisingadministering to a subject in need of such treatment or prevention (i) acomposition comprising an amount, effective for said treatment orprevention, of HSP and/or α2M complexed to a population of antigenicpeptides; and in combination with (ii) another treatment modality thatis a non-HSP and non-α2M-based treatment modality. The additionaltreatment modality is preferably a non-vaccine treatment modality.Examples of treatment modalities include but are not limited toantibiotics, antivirals, antifungal compounds, antiprotozoal compounds,antihelminth compounds, anti-cancer treatments such as chemotherapeuticagents, antiangiogenic compounds, hormones, and radiation, as well asbiological therapeutic agents and immunotherapeutic agents.

[0023] In another embodiment, a method is provided for treating orpreventing a type of cancer or infectious disease, comprisingadministering to a subject in need of such treatment or preventionantigen presenting cells which have been sensitized with complexes ofHSP and/or α2M and a population of antigenic proteins/peptides. Inaddition to the administration of sensitized antigen presenting cells toa subject, complexes of HSP and/or α2M and a population of antigenicpeptides; and/or a non-HSP and non-α2M-based treatment modality can alsobe administered to the subject.

[0024] The invention also provides methods for improving the therapeuticoutcome of a non-HSP and non-α2M-based treatment modality comprisingadministering either HSP complexes or α2M complexes, preferably purifiedcomplexes, in conjunction with the administration of the treatmentmodality.

[0025] In one embodiment of the invention, a method is provided forinducing an immune response in a subject against a first antigenic cellor viral particle, wherein said subject is receiving a non-HSP andnon-α2M treatment modality, said method comprising administering to theindividual a composition comprising an immunogenic amount of HSP and/orα2M complexed to a population of antigenic proteins/peptides that wereprepared from a second antigenic cell or viral particle. The antigenicpeptides can be obtained by digesting the protein preparation of theantigenic cells or viral particles with a protease or exposing theprotein preparation with ATP, guanidium hydrochloride and/or acidiccondition. The first and second antigenic cells or viral particlesexpress at least one common antigenic determinant.

[0026] In another embodiment, the present invention also provides amethod for improving the outcome of a treatment in a subject receivingHSP complexes or α2M complexes, by administering another therapeuticmodality to the subject before, concurrently with, or after theadministration of the HSP complexes or α2M complexes. Either the HSPcomplexes or the α2M complexes can be administered over a period of timewhich may precede, overlap, and/or follow a treatment regimen with anon-vaccine treatment modality.

[0027] The administering of the HSP complexes or α2M complexes to asubject can be repeated at the same site, and periodically, for example,at weekly intervals. The composition can be administered by many routes,such as intradermally or subcutaneously.

[0028] In yet another embodiment, the invention encompasses methods oftreatment that provide better therapeutic profiles than theadministration of the treatment modality or the HSP complexes alone. Inanother embodiment, the invention encompasses methods of treatment thatprovide better therapeutic profiles than the administration of thetreatment modality or the α2M complexes alone. Encompassed by theinvention are methods wherein the administration of a treatment modalitywith an HSP complexes or an α2M complexes has additive potency oradditive therapeutic effect. The invention also encompasses synergisticoutcomes where the therapeutic efficacy is greater than additive.Preferably, such administration of a treatment modality with an HSPcomplexes or with an α2M complexes also reduces or avoids unwanted oradverse effects.

[0029] Given the invention, in certain embodiments, doses of non-vaccinetreatment modality can be reduced or administered less frequently, inorder to increase patient compliance, improve therapy and/or reduceunwanted or adverse effects. In a specific embodiment, lower or lessfrequent doses of chemotherapy or radiation therapy are administered toreduce or avoid unwanted effects. Alternatively, doses of HSP complexesand doses of α2M complexes can be reduced or administered lessfrequently if administered with a treatment modality. In certainembodiments, the administration of the HSP/α2M complexes in the absenceof administration of the therapeutic modality or the administration ofthe therapeutic modality in the absence of administration of the HSP/α2Mcomplexes is not therapeutically effective. In a specific embodiment,the amount of HSP/α2M complexes or therapeutic modality is administeredin an amount insufficient to be therapeutically effective alone. Inalternate embodiments, both or at least one of the HSP/α2M complexes ortherapeutic modality is therapeutically effective when administeredalone.

4. DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention provides methods for preparing and using acomposition comprising heat shock protein (HSP) or alpha-2-macroglobulin(α2M) that are useful for the prevention and treatment of cancer andinfectious disease. The methods of the invention comprise preparing invitro complexes of HSP or α2M, and the antigenic proteins and peptidesof antigenic cells and using it in combination with another treatmentmodality. In one embodiment, the method involves making a proteinpreparation of the antigenic cells which preparation comprises apopulation of antigenic proteins; and complexing in vitro the populationof antigenic proteins to HSP or α2M. In another embodiment, the methodfurther involves digesting the protein preparation of the antigeniccells with at least one protease to generate a population of antigenicpeptides prior to complexing in vitro the population of antigenicpeptides to HSP or α2M. The invention exploits the full antigenicpotential of antigenic cells to generate a HSP- and/or α2M-basedvaccine.

[0031] The therapeutic and prophylactic methods of the invention arebased on eliciting an immune response in a subject in whom the treatmentor prevention of infectious diseases or cancer is desired, and who hasreceived or will receive another treatment modality. The immune responseis directed specifically against antigenic determinants of cancerouscells, cells infected by an infectious agent that causes the infectiousdisease, or antigenic determinants of the infectious agent. Byadministering to the individual a composition comprising a population ofmolecular complexes comprising HSPs and proteins/peptides of antigeniccells or a population of molecular complexes comprising α2M andproteins/peptides of antigenic cells, the molecular complexes in thecomposition can stimulate an immune response, such as a cytotoxic T cellresponse in the individual. The antigenic cells can be cancerous cellsor infected cells, or cells which share antigenic determinants with ordisplay similar antigenicity as the cancerous or infected cells. As aresult of the immune response, various immune effector mechanisms of theindividual will act on the cancerous or infected cells which can byitself or in combination with other treatment modalities lead to thetreatment or prevention of such disease.

[0032] The individual or subject in whom treatment or prevention of aninfectious diseases or cancer is desired is an animal, preferably amammal, a non-human primate, and most preferably human. The term“animal” as used herein includes but is not limited to companionanimals, such as cats and dogs; zoo animals; wild animals, includingdeers, foxes and racoons; farm animals, livestock and fowl, includinghorses, cattle, sheep, pigs, turkeys, ducks, and chickens, as well asany rodents.

[0033] The compositions and methods of the present invention are animprovement over other compositions and methods that usenaturally-occurring HSP-antigenic peptide complexes to treat or preventcancer or infectious disease. In such other methods, a specific HSP andits complexes with antigenic peptides are isolated from a cancer orinfected cell, and administered to a patient to induce an immuneresponse against the cancer or infected cells in vivo (see e.g., U.S.Pat. Nos. 5,750,119 and 5,961,979). Naturally-occurring complexes areisolated by methods dictated by the type of HSP which is desired. Thus,naturally-occurring complexes of a type of HSP and antigenic peptidescomprise only those antigenic peptides that are co-localized in acompartment of the antigenic cells with that type of HSP. Certain typesof HSPs are found uniquely in one cellular compartment and someantigenic peptides are found only in certain compartments of anantigenic cell. For example, HSP90 and HSP70 are found only in thecytosol. Thus, they will only be complexed to antigenic peptides locatedin the cytosol but not to antigenic peptides located somewhere else,such as the endoplasmic reticulum for example. That is, only a subset ofthe antigenic peptides of the antigenic cell can bind to each particularHSP. Thus, to stimulate an immune response to a maximum number ofantigenic determinants of a cancer or infected cell, every type of HSPsand their peptide complex would have to be isolated from the cancerousor infected cell by their respective methods of isolation, and thenadministered to a patient. This approach is laborious and may requirelarge amounts of antigenic cells which is not available under certaincircumstances. The methods of the present invention solve this problemby generating a peptide profile of virtually all the antigens of anantigenic cell in vitro, and then complexing the peptides to one or moredifferent HSP and/or α2M which can then be used to stimulate an immuneresponse in a patient. By using the methods of the invention, evenantigenic peptides and HSPs that are not co-localized within the samecompartment of an antigenic cell can form a complex. The methods of theinvention afford the possibility to form complexes of a particular typeof HSP with a majority of or even every antigenic peptides of anantigenic cell. Accordingly, a more effective immune response againstantigenic cells can be induced by the compositions prepared by themethods of the invention. Moreover, this method does not require theprior isolation of HSP complexes and the associated peptides, thus,allowing the use of very small amount of starting material which isoften limited in supply.

[0034] Moreover, the antigen profile of cancerous cells, infected cells,or pathogens may change over a period of time, e.g., even during acourse of treatment. Many pathogens evade the host immune system bymutation and synthesis of mutant proteins that are not recognized byimmune cells and antibodies. Cancerous cells are known to becomeresistant to certain drugs by mutations resulting in the synthesis ofmutant proteins, some of which may not be recognized by the immunesystem. An advantage of using one of the embodiments of the presentinvention is that by digesting the cytosolic and/or membrane-derivedproteins from cancerous cells, infected cells or pathogens, a widerrange of antigenic proteins and hence a greater diversity of antigenicpeptides are complexed to HSPs and/or α2M. As a result, the immuneresponse is directed to a greater number of antigenic determinants onthe antigenic cells, thus, making it more difficult for the antigeniccell, such as a cancer cell or an infected cell, to escape recognitionand action by the immune system.

[0035] In another specific embodiment, the methods of the presentinvention generate α2M-peptide complexes that are not found naturally.α2M is an extracellular protein that is known to bind to variousextracellular proteins, proteases in particular, to inactivate them andthen bring them to the intracellular environment. α2M does not generallyhave access and therefore does not complex to the entire repertoire ofantigenic peptides of an antigenic cell. The methods of the presentinvention allow α2M to be complexed to a much wider range of peptidesthat are cytosolic or membrane-derived, or that are generated by the invitro digestion of cytosolic and membrane-derived proteins of antigeniccells.

[0036] Described in Section 4.1 are sources of antigenic cells fromwhich protein preparations can be made. In Section 4.2, methods formaking different types of protein preparations of antigenic cells andmethods for digesting a protein preparation are provided. Section 4.3describes respectively the isolation or production of HSP or α2M, whichare used in complexing with antigenic peptides. The in vitro complexingof HSP and antigenic peptides are described in Section 4.4. Described inSection 4.5 are methods of use of the complexes in the prevention andtreatment of cancer and infectious agents, and the types of cancer andinfectious diseases that are treated. The use of the compositionsprepared by the methods of the invention in adoptive immunotherapy, istaught in Section 4.6. Section 5 provides experimental data showing theeffectiveness of the complexes of the invention in protecting an animalprophylactically from cancer cell growth.

4.1. Sources of Antigenic Cells

[0037] The antigenic cells of the invention comprise an antigenicdeterminant to which an immune response in a subject is desired.

[0038] For the treatment or prevention of cancer or infectious disease,the methods of the invention provide compositions of HSPs and α2Mcomplexed to antigenic proteins and peptides, which antigenicproteins/peptides were derived from cancer cells, preferably humancancers, e.g., fragments of tumor-specific antigens and tumor associatedantigens. The peptides can be generated by proteolytic digestion ofproteins (e.g., cytosolic and/or membrane-derived proteins) from cancercells, or antigenic cells that share antigenic determinants with ordisplay similar antigenicity as the cancer cells. The antigenic peptidescan also be generated by exposing the proteins to ATP, guanidiumhydrochloride, and/or acidic conditions. As used herein, the term “cellsor tissue of the same type of cancer” refers to cells or tissue ofcancer of the same tissue type, or metastasized from cancer of the sametissue type.

[0039] For the treatment or prevention of infectious diseases, themethods of the invention provide compositions of HSPs and α2M complexedto antigenic peptides that were derived from cells infected by apathogen or infectious agent that causes the infectious disease, or thepathogen which includes but is not limited to, a virus, bacterium,fungus, protozoan, parasite, etc. Preferably, the pathogen is one thatinfects humans. The antigenic peptides are generated by proteolyticdigestion of (e.g., cytosolic and/or membrane-derived) proteins obtainedfrom infected cells, antigenic cells that share antigenic determinantswith or display similar antigenicity as the infected cells, or thepathogens including viral particles. The antigenic peptides can also begenerated by exposing the proteins to ATP, guanidium hydrochloride,and/or acid. The antigenic peptides can also be generated from antigeniccells that display the antigenicity of an agent (pathogen) that causesthe infectious disease, or a variant of such agent.

[0040] Since whole cancer cells, infected cells or other antigenic cellsare used in the present methods, it is not necessary to isolate orcharacterize or even know the identities of these antigenic peptides inadvance of using the present methods. The source of the antigenic cellsmay be selected, depending on the nature of the disease with which theantigens are associated. In one embodiment of the invention, anytissues, or cells isolated from a cancer, including cancer that hasmetastasized to multiple sites, can be used as an antigenic cell in thepresent method. For example, leukemic cells circulating in blood, lymphor other body fluids can also be used, solid tumor tissue (e.g., primarytissue from a biopsy) can be used. As used herein, the term cancer cellalso encompasses a preneoplastic cell which is a cell in transition froma normal to a neoplastic form. The transition from non-neoplastic cellgrowth to neoplasia commonly consists of hyperplasia, metaplasia, anddysplasia (for review of such abnormal growth conditions (See Robbinsand Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co.,Philadelphia, pp. 68-79). A non-limiting list of cancers, the cells ofwhich can be used herein is provided in Section 4.5.1 below.

[0041] In another embodiment of the invention, any cell that is infectedwith a pathogen or infectious agent, i.e., an infected cell, can be usedas an antigenic cell for the preparation of antigenic peptides. Inparticular, cells infected by an intracellular pathogen, such as avirus, bacterium, fungus, parasite, or protozoan, is preferred. Anexemplary list of infectious agents that can infect cells which can beused herein is provided in Section 4.5.2.

[0042] In yet another embodiment, any pathogen or infectious agent thatcan cause an infectious disease can be used as antigenic cell for thepreparation of antigenic peptides. Variants of a pathogen or infectiousagent, such as but limited to replication-defective variants,non-pathogenic or attenuated variants, non-infectious variants, can alsobe used as an antigenic cell for this purpose. For example, manyviruses, bacteria, fungi, parasites and protozoans that can be culturedin vitro or isolated from infected materials can serve as a source ofantigenic cells. Methods known in the art for propagating such pathogensincluding viral particles can be used. An exemplary list of pathogens orinfectious agents that can be used as antigenic cells is provided inSection 4.5.2.

[0043] Cell lines derived from cancer tissues, cancer cells, or infectedcells can also be used as antigenic cells. Cancer or infected tissues,cells, or cell lines of human origin are preferred. Cancer cells,infected cells, or antigenic cells can be identified and isolated by anymethod known in the art. For example, cancer cells or infected cells canbe identified by morphology, enzyme assays, proliferation assays, or thepresence of pathogens or cancer-causing viruses. If the characteristicsof the antigens of interest are known, antigenic cells can also beidentified or isolated by any biochemical or immunological methods knownin the art. For example, cancer cells or infected cells can be isolatedby surgery, endoscopy, other biopsy techniques, isolation from bodyfluids (such as blood), affinity chromatography, and fluorescenceactivated cell sorting (e.g., with fluorescently tagged antibody againstan antigen express by the cells). Antigenic cells that display similarantigenicity have one or more antigenic determinants in common againstwhich an immune response in a subject is desired (e.g., for therapeuticor prophylactic purposes).

[0044] If the number of antigenic cells obtained from a subject isinsufficient, the cells may be cultured in vitro by standard methods toexpand the number of cells prior to use in the present methods. There isno requirement that a clonal or homogeneous or purified population ofantigenic cells be used. A mixture of cells can be used provided that asubstantial number of cells in the mixture contain the antigenicdeterminants or antigens of interest. In a specific embodiment, theantigenic cells and/or immune cells are purified.

[0045] In order to prepare pathogen-infected cells, uninfected cells ofa cell type susceptible to infection by the pathogen or infectious agentthat causes the disease can be infected in vitro. Depending on the modeof transmission and the biology of the pathogen or infectious agent,standard techniques can be used to facilitate infection by the pathogenor infectious agent, and propagation of the infected cells. For example,influenza viruses may be used to infect normal human fibroblasts; andmycobacteria may be used to infect normal human Schwann cells. Invarious embodiments, variants of an infectious agent, such asreplication-defective viruses, non-pathogenic or attenuated mutants, ortemperature-sensitive mutants can also be used to infect or transformcells to generate antigenic cells for the preparation of antigenicpeptides. If large numbers of a pathogen are needed to infect cells, orif pathogens are used directly as antigenic cells, any method known inthe art can be used to propagate and grow the pathogens. Such methodswill depend on the pathogen, and may not involve infecting a host. Forexample, many techniques are known in the art for growing pathogenicbacteria, fungi and other non-viral microorganisms in culture, includinglarge scale fermentation.

[0046] Alternatively, if the gene encoding a tumor antigen (e.g.,tumor-specific antigen and tumor-associated antigen) or antigen of thepathogen is available, normal cells of the appropriate cell type fromthe intended recipient may be transformed or transfected in vitro withan expression construct comprising a nucleic acid molecule encoding suchantigen, such that the antigen is expressed in the recipient's cells. Inone embodiment, a tumor-associated antigen is an antigen that isexpressed at a higher level in a tumor cell. relative to a normal cell;a tumor-specific antigen is an antigen that is expressed only in a tumorcell and not in a normal cell. Optionally, more than one such antigenmay be expressed in the recipient's cell in this fashion, as will beappreciated by those skilled in the art, any techniques known, such asthose described in Ausubel et al. (1989, Current Protocols in MolecularBiology, Wiley Interscience), may be used to perform the transformationor transfection and subsequent recombinant expression of the antigengene in recipient's cells.

[0047] Suitable proteins and peptides that may be expressed in suchcells include, but are not limited to those displaying the antigenicityof cancer cells. For example, such tumor specific or tumor-associatedantigens include but are not limited to KS 1/4 pan-carcinoma antigen(Perez and Walker, 1990, J. Immunol. 142:3662-3667; Bumal, 1988,Hybridoma 7(4):407-415); ovarian carcinoma antigen (CA125) (Yu, et al.,1991, Cancer Res. 51(2):468-475); prostatic acid phosphate (Tailer, etal., 1990, Nucl. Acids Res. 18(16):4928); prostate specific antigen(Henttu and Vihko, 1989, Biochem. Biophys. Res. Comm. 160(2):903-910;Israeli, et al., 1993, Cancer Res. 53:227-230); melanoma-associatedantigen p97 (Estin, et al, 1989, J. Natl. Cancer Inst. 81(6):445-446);melanoma antigen gp75 (Vijayasardahl, et al., 1990, J. Exp. Med.171(4):1375-1380); high molecular weight melanoma antigen (Natali, etal., 1987, Cancer 59:55-63), prostate specific membrane antigen,tyrosinase, gp 100, melan-A, and mucins. Other exogenous antigens thatmay be complexed to HSPs/α2M include portions or proteins that aremutated at a high frequency in cancer cells, such as oncogenes (e.g.,ras, in particular mutants of ras with activating mutations, which onlyoccur in four amino acid residues (12, 13, 59 or 61) (Gedde-Dahl et al.,1994, Eur. J. Immunol. 24(2):410-414)) and tumor suppressor genes (e.g.,p53, for which a variety of mutant or polymorphic p53 peptide antigenscapable of stimulating a cytotoxic T cell response have been identified(Gnjatic et al., 1995, Eur. J. Immunol. 25(6):1638-1642).

[0048] Preferably, where it is desired to treat or prevent viraldiseases, suitable proteins and peptides comprising epitopes of knownviruses can be expressed in the appropriate cells. For example, suchantigenic epitopes from viruses include, but not limited to, hepatitistype A, hepatitis type B, hepatitis type C, influenza, varicella,adenovirus, herpes simplex type I (HSV-I), herpes simplex type II(HSV-II), rinderpest, rhinovirus, echovirus, rotavirus, respiratorysyncytial virus, papilloma virus, papova virus, cytomegalovirus,echinovirus, arbovirus, huntavirus, coxsackie virus, mumps virus,measles virus, smallpox virus, rubella virus, polio virus, humanimmunodeficiency virus type I (HIV-I), and human immunodeficiency virustype II (HIV-II).

[0049] Preferably, where it is desired to treat or prevent bacterialinfections, suitable proteins and peptides comprising epitopes of knownbacteria can be expressed in the appropriate cells. For example, suchbacterial epitopes may be derived from various bacteria including, butnot limited to, Gram positive bacillus (e.g., Listeria, Bacillus such asBacillus anthracis, Erysipelothrix species), Gram negative bacillus(e.g., Bartonella, Brucella, Campylobacter, Enterobacter, Escherichia,Francisella, Hemophilus, Klebsiella, Morganella, Proteus, Providencia,Pseudomonas, Salmonella, Serratia, Shigella, Vibrio, and Yersiniaspecies), spirochete bacteria (e.g., Borrelia species including Borreliaburgdorferi that causes Lyme disease, and Leptospira), anaerobicbacteria (e.g., Actinomyces and Clostridium species including C. tetani,C. botulinum, C. perfringens), Gram positive and negative coccalbacteria, Streptococcus species, Pneumococcus species, Staphylococcusspecies (e.g., S. aureus and S. pneumonia), Neisseria species (e.g., N.meningitidis).

[0050] Preferably, where it is desired to treat or prevent fungalinfections, suitable proteins and peptides comprising epitopes of knownfungi can be expressed in the appropriate cells. For example, suchantigenic epitopes may be derived from various fungi including,Aspergillus (e.g., Aspergillus fumigatus), Cryptococcus (e.g.,Cryptococcus neoformans), Sporotrix, Coccidioides, Paracoccidioides,Histoplasma, Blastomyces, Candida (e.g., Candida albicans), Rhizopus,Rhizomucor, Absidia, and Basidiobolus species.

[0051] Preferably, where it is desired to treat or prevent parasiticinfections, suitable proteins and peptides comprising epitopes of knownprotozoa, nematodes, or helminths can be expressed in the appropriatecells. For example, such antigenic epitopes may be derived from variousprotozoa including, but not limited to, Entoamoeba, Plasmodium,Leishmania, Eimeria, Cryptosporidium, Giardiasis, Toxoplasma, andTrypanosoma species.

4.2. Preparation of Antigenic Proteims and Peptides

[0052] According to the invention, the compositions of the inventioncomprise antigenic proteins complexed to HSPs, wherein the antigenicproteins are from a preparation of proteins of the antigenic cells ofinterest. The compositions of the invention also comprise antigenicproteins complexed to α2M, wherein the antigenic proteins are from apreparation of proteins of the antigenic cells of interest. Thecompositions of the invention also comprise complexes of HSPs andantigenic peptides, or complexes of α2M and antigenic peptides that areprepared by first, generating a population of peptides from apreparation of proteins of the antigenic cells of interest, and thencomplexing the peptides to HSPs or α2M.

[0053] In various embodiments, to maximize and preserve the diversity ofantigenic proteins and peptides, the methods used for preparing aprotein preparation of antigenic cells do not selectively remove orretain any particular protein or peptide from other proteins andpeptides in the antigenic cell. Even in certain embodiments whencytosolic proteins or membrane-derived proteins are used, the methodsused to make the preparations do not selectively remove or retain anyparticular protein of the cytosol or of the membranes. Therefore, themajority of the proteins present in the cytosol or in the membranes arealso present in the respective preparations of antigenic proteins andpeptides from antigenic cells. In preferred embodiments, substantiallythe entire repertoire of antigenic proteins and peptides of theantigenic cells, and substantially all the antigenic proteins andpeptides in the cytosol or in the membranes are present in thecomplexing reaction and form complexes with HSPs and/or α2M.

[0054] 4.2.1 Protein Preparations of Antigenic Cells

[0055] In one embodiment of the invention, a protein preparation isprovided which is derived from a cancer cell, infected cell, orpathogen. For example, for the treatment of cancer, the proteinpreparations are prepared, postoperatively, from tumor cells obtainedfrom a cancer patient. In another embodiment of the present invention,one or more antigenic proteins of interest are synthesized in cell linesmodified by the introduction of recombinant expression systems thatencode such antigens, and such cells are used to prepare the proteins.The proteins can be obtained from one or more cellular fraction(s), forexample, the cytosol of the antigenic cells, or they can be extracted orsolubilized from the membranes or cell walls of the antigenic cells. Anytechnique known in the art for cell lysis, fractionation of cellularcontents, and protein enrichment or isolation can be used. See, forexample, Current Protocols in Immunology, vol. 2, chapter 8, Coligan etal. (ed.), John Wiley & Sons, Inc.; Pathogenic and ClinicalMicrobiology: A Laboratory Manual by Rowland et al., Little Brown & Co.,June 1994; which are incorporated herein by reference in theirentireties. Depending on the techniques used to fractionate the cellularcontents, a cellular fraction comprises at least 20, 50, 100, 500,1,000, 5,000, 10,000, or 20,000 different proteins.

[0056] As used herein, the term “protein preparation” refers to amixture of proteins obtained from antigenic cells, a cellular fractionof antigenic cells, or virus particles. The proteins can be obtainedfrom a cellular fraction, such as the cytosol. The proteins can also benon-cytosolic proteins (e.g., those from cell walls, cell membranes ororganelles), or both. Cellular fractions may include but are not limitedto cytosolic fractions, membrane fractions, and organelle fractions,such as nuclear, mitochondrial, lysosomal, and endoplasmicreticulum-derived fractions. The protein preparations can be obtainedfrom non-recombinant or recombinant cells. The term “antigenic proteins”as used herein also encompasses antigenic polypeptides and antigenicpeptides that may be present in the preparation. The protein preparationobtained from the antigenic cells or cellular fractions thereof or virusparticles can optionally be purified from other non-proteinaceousmaterials to various degrees by techniques known in the art. The proteinpreparation may comprise at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95% 97%, 98%, 99% of the different proteins and peptidespresent in the antigenic cells or virus particles or a fraction of theantigenic cells.

[0057] In a specific embodiment, the protein preparations have not beensubjected to any method of preparation that selectively removes orretains one or more particular protein(s) from the other proteins in theantigenic cells.

[0058] In a specific embodiment, the protein preparation is the totalcell lysate which is not fractionated and/or purified, and may containother non-proteinaceous materials of the cells.

[0059] In another specific embodiment, the protein preparation is thetotal protein in a cellular fraction, which has not been subjected tofurther fractionation or purification, and may contain othernon-proteinaceous materials of the cells.

[0060] In yet another embodiment, the protein preparation is the totalprotein in a preparation of viral particles.

[0061] In specific embodiments, the protein preparation comprises totalcellular protein, total cytosolic proteins, or total membrane-boundproteins of antigenic cell(s).

[0062] In various embodiments, the protein preparation comprises atleast 20, 50, 100, 500, 1,000, 5,000, 10,000, or 20,000 differentproteins. A plurality of different antigenic proteins are present in aprotein preparation of antigenic cells. Moreover, the proteins in theprotein preparation may be subjected to a step of protease digestionprior to in vitro complexing to HSPs or α2M. Alternatively, the proteinsin the protein preparation are not subjected to a step of proteasedigestion prior to in vitro complexing to HSPs or α2M.

[0063] To make a protein preparation of antigenic cells or virusparticles, the lysing of antigenic cells or disruption of cell walls,cell membranes, or viral particle structure can be performed usingstandard protocols known in the art. In various embodiments, theantigenic cells can be lysed, for example, by mechanical shearing,sonication, freezing and thawing, adjusting the osmolarity of the mediumsurrounding the cells, or a combination of techniques. In less preferredembodiments, the antigenic cells can be lysed by chemicals, such asdetergents.

[0064] Once the cells are lysed, it is desirable to remove cellulardebris, materials that are non-proteinaceous or materials that do notcontain cytosolic, and/or membrane-derived proteins (including proteinsin the membranes of organelles). Removal of these components can beaccomplished by techniques such as low speed centrifugation orfiltration. After removing cellular debris and intact cells, a highspeed centrifugation step can be used to separate the cytosolic proteinswhich are in the supernatant, and the membrane-derived proteins whichare collected in the pellet. Standard procedures commonly known in theart allows the further isolation of the membrane-derived proteins fromthe pellet. Standard techniques commonly known in the art can be used toextract viral proteins from viral particles. These separation methodsact on the basis of the general and overall size, density, and/or chargeof the molecules that are present in the antigenic cell, in the cytosolor in the membranes. These separation methods do not or are not designedto selectively remove or retain any one or more particular protein(s)from other proteins.

[0065] In various embodiments, the proteins from the antigenic cells canbe optionally separated by their general biochemical and/or biophysicalproperties, such as size, density, charge, cellular location orcombinations thereof. Many techniques known in the art can be used toperform the separation. Selected fractions of the proteins/peptides thatcomprise at least 20, 50, 100, 500, 1,000, 5,000, 10,000, or 20,000different proteins or that comprise at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95% 97%, 98%, 99% of the different proteins presentin the antigenic cells or a cellular fraction thereof, or virusparticles, can be used to form complexes to HSP or α2M. Accordingly, theproteins from the antigenic cells can be prepared by methods thatseparate molecules by their size, charge, cellular location or acombination thereof, and that do not selectively remove or retain anyone or more specific protein(s) from other proteins that are present inthe antigenic cell, in the cytosol or in the membranes.

[0066] An exemplary, but not limiting, method that may be used to make aprotein preparation comprising cytosolic proteins is as follows:

[0067] Cells, which may be tumor cells derived from a biopsy of thepatient or tumor cells cultivated in vitro, or cell infected with apathogenic agent, are suspended in 3 volumes of 1× Lysis buffercomprising 30 mM sodium bicarbonate pH 7.5, 1 mM PMSF, incubated on icefor 20 minutes and then the hypotonically-swollen cells are homogenizedin a dounce homogenizer until >95% cells are lysed. As an alternative toshearing, cells can be sonicated, on ice, until >99% cells are lysed asdetermined by microscopic examination. When sonication is used, cellsare suspended in a buffer such as phosphate buffered saline (PBS) whichmay comprises 1 mM PMSF, before sonication.

[0068] The lysate is centrifuged at 1,000×g for 10 minutes to removeintact cells, nuclei and other cellular debris. The resultingsupernatant is recentrifuged at about 100,000×g for about one hour, andthe supernatant recovered. The 100,000×g supernatant may be dialyzed for36 hours at 4° C. (three times, 100 times volumes each time) against PBSor other suitable buffer, to provide the soluble cytosolic proteins ofthe present invention. If necessary, insoluble material in thepreparation may be removed by filtration or low-speed centrifugation.

[0069] An exemplary, but not limiting, method that may be used to make aprotein preparation comprising membrane-derived proteins is as follows:

[0070] Cells, which may be tumor cells derived from a biopsy of thepatient or tumor cells cultivated in vitro, or cells infected with apathogenic agent, are suspended in 3 volumes of 1× Lysis buffercomprising 30 mM sodium bicarbonate pH 7.5, 1 mM PMSF, incubated on icefor 20 minutes and then the hypotonically-swollen cells are homogenizedin a dounce homogenizer until >95% cells are lysed. As an alternative toshearing, cells can be sonicated, on ice, until >99% cells are lysed asdetermined by microscopic examination. When sonication is used, cellsare suspended in a buffer such as phosphate buffered saline (PBS) whichmay comprises 1 mM PMSF, before sonication.

[0071] The lysate is then centrifuged at 100,000×g for 10 minutes tocollect the cell membranes. Membrane-derived proteins can be dislodgedfrom the lipid bilayer and isolated from the 100,000 g pellet (where themembrane-derived proteins are located) by resuspending the pellet in 5volumes of PBS containing 1% sodium deoxycholate (without Ca²⁺ and Mg²⁺)and incubated on ice for 1 h. The resulting suspension is centrifugedfor 30 min at 20,000 g and the resulting supernatant harvested anddialyzed against several changes of PBS (without Ca²⁺ and Mg²⁺) toremove the detergent. The resulting dialysate is centrifuged for 90 minat 100,000 g and the supernatant purified further. Then calcium andmagnesium are both added to the supernatant to give final concentrationsof 2 mM. If necessary, insoluble material in the preparation may beremoved by filtration or low-speed centrifugation.

[0072] In a specific embodiment, the population of cytosolic and/ormembrane-derived proteins obtained from antigenic cells can be complexedto HSP or α2M directly without protease treatment or any furtherextraction or selection processes. Alternatively, the proteins can besubjected to protease treatment prior to complexing.

[0073] 4.2.2 Peptides from Antigenic Cells

[0074] According to the invention, the cytosolic and membrane-derivedproteins obtained from antigenic cells can be optionally digested togenerate antigenic peptides. In one embodiment, either the cytosolic orthe membrane-derived proteins are used in the digestion. In anotherembodiment, the cytosolic and membrane-derived proteins are combined inthe digestion reaction to generate antigenic peptides. In preferredembodiments, the protein preparations that are used in the proteasedigestion have not been subjected to any method(s) of preparation thatselectively remove or retain one or more particular protein(s) from theother proteins in the antigenic cells, or the cytosol or membranes ofthe antigenic cells.

[0075] Various proteases or proteolytic enzymes can be used in theinvention to produce from a protein preparation of antigenic cells apopulation of peptides which comprises antigenic peptides. The enzymaticdigestions can be performed either individually or in suitablecombinations with any of the proteolytic enzymes that are well known inthe art including, but not limited to, trypsin, Staphylococcal peptidaseI (also known as protease V8), chymotrypsin, pepsin, cathepsin G,thermolysin, elastase, and papain. Trypsin is a highly specific serineproteinase that cleaves on the carboxyl-terminal side of lysines andarginines. Due to the limited number of cleavage sites, it is expectedto leave many MHC-binding epitopes intact. Staphylococcal peptidase I, aserine proteinase, has specificity for cleavage after glutamic andaspartic acid residues. A digestion can be carried out with a singleprotease or a mixture of proteases. The proteases or proteolytic enzymesused are incubated under conditions suitable for the particular enzyme.Preferably, the enzyme is purified. Non-enzymatic methods, such ascyanogen bromide cleavage, can also be used for generating peptides. Theprotein preparation to be digested can be aliquoted into a plurality ofreactions each using a different enzyme, and the resulting peptides mayoptionally be pooled together for use. It may not be necessary tocompletely digest the proteins in the enzymatic reactions. Thesereactions results in the generation of a diverse and different set ofpeptides for each protein that is present in the protein preparation.The production of different peptide sets allows for a greaterprobability of generating antigenic peptides that are capable ofinducing an immune response to the antigens in the protein preparationwhen they are complexed to HSP or α2M. In a preferred embodiment, theprotein preparation to be digested is aliquoted into two separatereactions and two different proteolytic enzymes are used to produce twodifferent sets of peptides of the proteins present in the proteinpreparation. Depending on the proteins, enzymes and reaction conditions,undigested proteins may remain in the reactions. In a preferredembodiment, trypsin and Staphylococcal peptidase I are used separatelyto digest the protein preparation.

[0076] In another preferred embodiment, the proteolytic enzymes used inthe invention exhibit similar activities as the proteolytic activitiesthat are found in the proteasome. The proteasome is responsible for theextralysosomal, endocatalytic degradation of cytosolic and nuclearproteins which are mis-folded or damaged in a cell. The proteasome candegrade proteins completely to yield single amino acids, can generateoptimal major histocompatibility complex class I (MHC I)-bindingepitopes, and can generate longer peptide precursors which couldpotentially undergo further trimming elsewhere in the cell to yieldcytotoxic T cell epitopes. Cleavage preferences of the proteasome is onthe carboxyl (COOH)-side of basic, acidic, and hydrophobic amino acids.Three known proteolytic enzymatic activities that are present in theproteasome are chymotrypsin-like activity, trypsin-like activity, andpeptidylglutamylpeptide-hydrolyzing activity (Uebel and Tampe, 1999,Curr. Opin. Immunol. 11:2 203-208). As such, enzymes having suchactivities and specificities can be used separately or in combination todigest the protein preparation. In a preferred embodiment, trypsin,chymotrypsin, and/or peptidylglutamylpeptide-hydrolase are used.

[0077] The resulting peptide digestions comprise antigenic peptides,non-antigenic peptides, and single amino acid residues. The reactionsmay also comprise undigested or incompletely digested antigenicproteins. The proteolytic enzymatic digestions of the invention aremonitored in order to generate peptides that fall within a desirablerange of lengths. In a preferred embodiment, the peptides generated arefrom about 7 to about 20 amino acid residues. Most antigenic peptidesthat are presented to T cells by MHC class I and class II fall withinthis range. In various embodiments, the population of peptides comprisespeptides having a size range of 6 to 21, 8 to 19, 10 to 20, or at least7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 40, 45, or 50, amino acid residues.In preferred embodiments, the antigenic peptides have 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues. To monitorthe progression of protein digestion, a test reaction can be performedwhere small aliquots of a protein digestion are taken out of thereaction and monitored for the progression of digestion through eithertricine-polyacrylamide gel electrophoresis (“tricine-PAGE”), highperformance liquid chromatography (“HPLC”), or mass spectrometry, or anyother method known in the art to determine the size of peptides. Usingsuch a test reaction, a determination can be made as to when peptidefragments of a particular size range will be generated at a particularenzymatic concentration. Other variables of the reaction that can bemanipulated include the amount of protein in the reaction, thetemperature, the duration of incubation, the presence of cofactors, etc.

[0078] Once the proper conditions are established for the generation ofpeptide fragments of a particular size range from a type of antigeniccell, the enzymatic reaction conditions can be duplicated to generateantigenic peptides which can be pooled. It is preferred that theenzymatic digestion is terminated before the peptides are complexed toHSPs or α2M. In one embodiment of the invention, inhibitors can be usedfor terminating an enzymatic digestion. Enzymatic inhibitors that can beused in the invention include, but are not limited to, PMSF, bestatin,amastatin, leupeptin, and cystatin, depending on which enzymes are usedin the protein digestion. Inhibitors for most proteases are well knownin the art. Alternatively, another method of terminating an enzymaticdigestion is by physical removal of the enzyme from the reaction. Thiscan be done by attaching the enzyme of choice to a solid phase, such asa resin or a material that can easily be removed from the reaction bywell known methods such as centrifugation or filtration. The proteinpreparation is allowed to contact or flow across the solid phase for aperiod of time. Such immobilized enzymes can be purchased commerciallyor can be produced by procedures for immobilizing enzymes that are wellknown in the art.

[0079] At the end of the digestion reaction, the peptides can optionallybe separated from low molecular weight materials, such as dipeptides, orsingle amino acid residues, in the preparation. For example, thepeptides can be isolated by centrifugation through a membrane, such asthe Centriprep-3. Optionally, the peptides can be separated by theirgeneral biochemical and/or biophysical properties, such as size, charge,or combinations thereof. Any techniques known in the art can be used toperform the separation resulting in digested protein preparationcomprising at least 50, 100, 500, 1,000, 5,000, 10,000, 20,000, 50,000,or 100,000 different peptides.

[0080] In another embodiment of the invention, peptides that areendogenously present in antigenic cells can be used in the inventioneither alone or in combination with the peptides generated by theproteolytic digestion of the cytosolic and membrane-derived proteins.Peptides that are endogenously present in antigenic cells includepeptides that are complexed in vivo to HSP and/or MHC class I and IImolecules. According to the invention, such peptides that are isolateddirectly from a protein preparation of antigenic cells can be complexedto HSPs and/or α2M.

[0081] In specific embodiments, either the cytosolic or themembrane-derived proteins are used in the isolation process. In anotherspecific embodiment, the cytosolic and membrane-derived proteins arecombined in the isolation process. In preferred embodiments, the proteinpreparations that are used in the isolation have not been subjected toany method(s) of preparation that selectively remove or retain one ormore particular protein(s) from the other proteins in the antigeniccells, or the cytosol or membranes of the antigenic cells. The antigenicpeptides are isolated directly from a protein preparation of the cellwithout isolating complexes of antigenic peptides and HSP, α2M or majorhistocompatibility complex (MHC) molecules first. Preferably, theprotein preparation comprises comprise at least 20, 50, 100, 500, 1,000,5,000, 10,000, or 20,000 different proteins or that comprise at least50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% 97%, 98%, 99% of thedifferent proteins present in the antigenic cells or a cellular fractionthereof, or virus particles.

[0082] In various embodiments, the method comprise treating the proteinpreparation to ATP, guanidium hydrochloride, and/or exposing the proteinpreparation to acidic conditions such that antigenic peptides that areassociated with proteins such as HSPs, α2M, and MHC class I and IImolecules in the protein preparation can be eluted. Preferably, theisolation process does not comprise purifying HSP complexes, α2Mcomplexes, or MHC complexes for the protein preparation prior totreatment with ATP, guanidium hydrochloride, or acidic conditions. Manydifferent acids can be used, including but not limited to,trifluoroacetic acid. Methods are known in the art for the isolation ofpeptides from HSP-peptide complexes, such as Menoret et al., 1999,Biochem. Biophys. Res. Commun. 262(3):813-8, which is incorporatedherein by reference in its entirety. Methods known in the art such asthose described in Marston and Hartley (1990, Meth. Enzymol.182:264-276) for dissociating protein aggregates can also be used.

[0083] In particular, the isolation process comprises exposing a proteinpreparation of antigenic cells with ATP, for example, at roomtemperature for one hour, and/or treating a protein preparation ofantigenic cells with trifluoroacetic acid (TFA) at a concentration inthe range of 0.05% to 1% TFA. The treatment preferably comprisessonicating the protein preparation in the presence of 0.1% TFA. In amost preferred embodiment, a protein preparation is first exposed toATP, followed by sonication in 0.1% TFA. Various protease inhibitors canbe used in the invention prior to cell lysis and the isolation processto prevent or reduce cleavage of cellular protein that may generatepeptides that are not endogenously associated with HSPs or or α2M. Forexample, a mixture of 14 protease inhibitors can be used:phenylmethylsulfonyl fluoride (PMSF) 2 mM, ethylenediaminetetreacedicacid (EDTA) 1 mM, ethylene glycolbis(P-aminoethylether)N,N,N′,N′-tetraacetic acid (EGTA) 1 mM, (all obtained from Sigma,St. Louis, Mo.), and Antipain 20 mg/ml, Bestatin 5 mg/ml, Chemostatin 20ptg/nil, E64 20 Jig/ml, Leupeptine 1 ttg/ml, Pepstatine 1 gg/ml,Pefabloc 40 Ag/ml, and Apoprotein 10 tkg/rnl (all obtained fromBoehringer Mannheim, Indianapolis, Ind.). The peptides resulting fromthe protein preparation comprise antigenic peptides and non-antigenicpeptides of a variety of sizes ranging from at least 7, 8, 9, 10, 11,12, 15, 20, 25, 30, 40, 45, or 50, amino acid residues. At the end ofthe process, the peptides are preferably recovered by separating fromthe proteins in the preparation prior to complexing with HSP or α2M. Forexample, the peptides can be recovered by centrifugation through amembrane, such as the Centriprep-3, by drying under vacuum, or byreverse phase chromatography, e.g., fractionation in a BioCad20microanalytiocal HPLC Poros RH2 column (Perseptive Biosystems,Cambridge, Mass.), equilibrated with 0.1% TFA in water and elution byacetonitrile. Accordingly, antigenic peptides that are endogenouslypresent in antigenic cells and that are isolated directly from a proteinpreparation can be complexed to HSPs and/or α2M. Alternatively, a mixedpopulation of peptides comprising peptides that are endogenously presentin antigenic cells and peptides from digested cytosolic andmembrane-derived proteins, can be complexed to HSPs and/or α2M.

4.3. Preparation of HSPs and α2M

[0084] According to the present invention, antigenic peptides derivedfrom antigenic cells are complexed to HSPs and/or α2M. Described hereinare exemplary methods that can be used for isolating and preparing HSPsand α2M for use in the invention.

[0085] Heat shock proteins, which are also referred to interchangeablyherein as stress proteins, useful in the practice of the instantinvention can be selected from among any cellular protein that satisfiesthe following criteria. It is a protein whose intracellularconcentration increases when a cell is exposed to a stressful stimuli,it is capable of binding other proteins or peptides, it is capable ofreleasing the bound proteins or peptides in the presence of adenosinetriphosphate (ATP) or under acidic conditions; and it is a proteinshowing at least 35% homology with any cellular protein having the aboveproperties.

[0086] The first stress proteins to be identified were the heat shockproteins (HSPs). As their name implies, HSPs are synthesized by a cellin response to heat shock. To date, five major classes of HSPs have beenidentified, based on the molecular weight of the family members. Theseclasses are called sHSPs (small heat shock proteins), HSP60, HSP70,HSP90, and HSP 100, where the numbers reflect the approximate molecularweight of the HSPs in kilodaltons. In addition to the major HSPfamilies, an endoplasmic reticulum resident protein, calreticulin, hasalso been identified as yet another heat shock protein useful foreliciting an immune response when complexed to antigenic molecules (Basuand Srivastava, 1999, J. Exp. Med. 189:797-202). Other stress proteinsthat can be used in the invention include but are not limited to grp78(or BiP), protein disulphide isomerase (PDI), HSP110, and grp 170 (Linet al., 1993, Mol. Biol. Cell, 4:1109-1119; Wang et al., 2001, J.Immunol., 165:490-497). Many members of these families were foundsubsequently to be induced in response to other stressful stimuliincluding, but not limited to, nutrient deprivation, metabolicdisruption, oxygen radicals, hypoxia and infection with intracellularpathogens. (See Welch, May 1993, Scientific American 56-64; Young, 1990,Annu. Rev. Immunol. 8:401-420; Craig, 1993, Science 260:1902-1903;Gething, et al., 1992, Nature 355:33-45; and Lindquist, et al., 1988,Annu. Rev. Genetics 22:631-677), the disclosures of which areincorporated herein by reference. It is contemplated that HSPs/stressproteins belonging to all of these families can be used in the practiceof the instant invention.

[0087] The major HSPs can accumulate to very high levels in stressedcells, but they occur at low to moderate levels in cells that have notbeen stressed. For example, the highly inducible mammalian HSP70 ishardly detectable at normal temperatures but becomes one of the mostactively synthesized proteins in the cell upon heat shock (Welch, etal., 1985, J. Cell. Biol. 101:1198-1211). In contrast, HSP90 and HSP60proteins are abundant at normal temperatures in most, but not all,mammalian cells and are further induced by heat (Lai, et al., 1984, Mol.Cell. Biol. 4:2802-10; van Bergen en Henegouwen, et al., 1987, GenesDev. 1:525-31).

[0088] Heat shock proteins are among the most highly conserved proteinsin existence. For example, DnaK, the HSP70 from E. coli has about 50%amino acid sequence identity with HSP70 proteins from excoriates(Bardwell, et al., 1984, Proc. Natl. Acad. Sci. 81:848-852). The HSP60and HSP90 families also show similarly high levels of intrafamiliesconservation (Hickey, et al., 1989, Mol. Cell. Biol. 9:2615-2626;Jindal, 1989, Mol. Cell. Biol. 9:2279-2283). In addition, it has beendiscovered that the HSP60, HSP70 and HSP90 families are composed ofproteins that are related to the stress proteins in sequence, forexample, having greater than 35% amino acid identity, but whoseexpression levels are not altered by stress. Therefore it iscontemplated that the definition of heat shock protein or stressprotein, as used herein, embraces other proteins, muteins, analogs, andvariants thereof having at least 35% to 55%, preferably 55% to 75%, andmost preferably 75% to 85% amino acid identity with members of the threefamilies whose expression levels in a cell are enhanced in response to astressful stimulus.

[0089] In an embodiment wherein the HSP portion of the HSP-antigenicpeptide complex is desired to be purified from cells, exemplarypurification procedures such as described in Sections 4.3.1-4.3.3 belowcan be employed to purify HSP-peptide complexes, after which the HSPscan be separated from the endogenous HSP-peptide complexes in thepresence of ATP or under acidic conditions, for subsequent in vitrocomplexing to a population of antigenic peptides. See Peng, et al.,1997, J. Immunol. Methods, 204:13-21; Li and Srivastava, 1993, EMBO J.12:3143-3151, which are incorporated herein by reference in theiractivities. Although described for tumor cells, the protocols describedhereinbelow may be used to isolate HSPs from any infected cells, and anyeukaryotic cells, for example, tissues, isolated cells, or immortalizedeukaryote cell lines infected with an intracellular pathogen, tumorcells or tumor cell lines.

[0090] 4.3.1. Preparation and Purification of HSP70-Peptide Complexes

[0091] The purification of HSP70-peptide complexes has been describedpreviously, see, for example, Udono et al., 1993, J. Exp. Med.178:1391-1396. A procedure that may be used, presented by way of examplebut not limitation, is described below.

[0092] Initially, tumor cells are suspended in 3 volumes of 1× Lysisbuffer consisting of 30 mM sodium bicarbonate pH 7.5, 1 mM PMSF. Then,the pellet is sonicated, on ice, until >99% cells are lysed asdetermined by microscopic examination. As an alternative to sonication,the cells may be lysed by mechanical shearing by homogenizing the cellsin a Dounce homogenizer until >95% cells are lysed.

[0093] Then the lysate is centrifuged at 1,000 g for 10 minutes toremove unbroken cells, nuclei and other cellular debris. The resultingsupernatant is recentrifuged at 100,000 g for 90 minutes, thesupernatant harvested and then mixed with Con A Sepharose equilibratedwith phosphate buffered saline (PBS) containing 2 mM Ca²⁺ and 2 mM Mg²⁺.When the cells are lysed by mechanical shearing the supernatant isdiluted with an equal volume of 2× lysis buffer prior to mixing with ConA Sepharose. The supernatant is then allowed to bind to the Con ASepharose for 2-3 hours at 4° C. The material that fails to bind isharvested and dialyzed for 36 hours (three times, 100 volumes each time)against 10 mM Tris-Acetate pH 7.5, 0.1 mM EDTA, 10 mM NaCl, 1 mM PMSF.Then the dialyzate is centrifuged at 17,000 rpm (Sorvall SS34 rotor) for20 minutes. Then the resulting supernatant is harvested and applied to aMono Q FPLC column equilibrated in 20 mM Tris-Acetate pH 7.5, 20 mMNaCl, 0.1 mM EDTA and 15 mM 2-mercaptoethanol. The column is thendeveloped with a 20 mM to 500 mM NaCl gradient and then eluted fractionsfractionated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and characterized by immunoblotting using anappropriate anti-HSP70 antibody (such as from clone N27F3-4, fromStressGen).

[0094] Fractions strongly immunoreactive with the anti-HSP70 antibodyare pooled and the HSP70-peptide complexes precipitated with ammoniumsulfate; specifically with a 50%-70% ammonium sulfate cut. The resultingprecipitate is then harvested by centrifugation at 17,000 rpm (SS34Sorvall rotor) and washed with 70% ammonium sulfate. The washedprecipitate is then solubilized and any residual ammonium sulfateremoved by gel filtration on a Sephadex^(R) G25 column (Pharmacia). Ifnecessary the HSP70 preparation thus obtained can be repurified throughthe Mono Q FPLC Column as described above.

[0095] The HSP70-peptide complex can be purified to apparent homogeneityusing this method. Typically 1 mg of HSP70-peptide complex can bepurified from 1 g of cells/tissue.

[0096] An improved method for purification of HSP70 comprises contactingcellular proteins with ATP or a nonhydrolyzable analog of ATP affixed toa solid substrate, such that HSP70 in the lysate can bind to the ATP ornonhydrolyzable ATP analog, and eluting the bound HSP70. A preferredmethod uses column chromatography with ATP affixed to a solid substratum(e.g., ATP-agarose). The resulting HSP70 preparations are higher inpurity and devoid of contaminating peptides. The HSP70 yields are alsoincreased significantly by about more than 10 fold.

[0097] Alternatively, chromatography with nonhydrolyzable analogs ofADP, instead of ATP, can be used for purification of HSP70-peptidecomplexes. By way of example but not limitation, purification of HSP70free of peptide by ATP-agarose chromatography can be carried out asfollows:

[0098] Meth A sarcoma cells (500 million cells) are homogenized inhypotonic buffer and the lysate is centrifuged at 100,000 g for 90minutes at 4° C. The supernatant is applied to an ATP-agarose column.The column is washed in buffer and is eluted with 5 column volumes of 3mM ATP. The HSP70 elutes in fractions 2 through 10 of the total 15fractions which elute. The eluted fractions are analyzed by SDS-PAGE.The HSP70 can be purified to apparent homogeneity using this procedure.

[0099] 4.3.2. Preparation and Purification of HSP90-Peptide Complexes

[0100] A procedure that can be used, presented by way of example but notlimitation, is described below.

[0101] Initially, tumor cells are suspended in 3 volumes of 1× Lysisbuffer consisting of 30 mM sodium bicarbonate pH 7.5, 1 mM PMSF. Then,the pellet is sonicated, on ice, until >99% cells are lysed asdetermined by microscopic examination. As an alternative to sonication,the cells may be lysed by mechanical shearing by homogenizing the cellsin a Dounce homogenizer until >95% cells are lysed.

[0102] Then the lysate is centrifuged at 1,000 g for 10 minutes toremove unbroken cells, nuclei and other cellular debris. The resultingsupernatant is recentrifuged at 100,000 g for 90 minutes, thesupernatant harvested and then mixed with Con A Sepharose equilibratedwith PBS containing 2 mM Ca²⁺ and 2 mM Mg²⁺. When the cells are lysed bymechanical shearing the supernatant is diluted with an equal volume of2× Lysis buffer prior to mixing with Con A Sepharose. The supernatant isthen allowed to bind to the Con A Sepharose for 2-3 hours at 4° C. Thematerial that fails to bind is harvested and dialyzed for 36 hours(three times, 100 volumes each time) against 20 mM sodium phosphate pH7.4, 1 mM EDTA, 250 mM NaCl. Then the dialyzate is centrifuged at 17,000rpm (Sorvall SS34 rotor) for 20 minutes. Then the resulting supernatantis harvested and applied to a Mono Q FPLC column equilibrated withdialysis buffer. The proteins are then eluted with a salt gradient of200 mM to 600 mM NaCl.

[0103] The eluted fractions are fractionated by SDS-PAGE and fractionscontaining the HSP90-peptide complexes identified by immunoblottingusing an anti-HSP90 antibody such as 3G3 (Affinity Bioreagents).HSP90-peptide complexes can be purified to apparent homogeneity usingthis procedure. Typically, 150-200 μg of HSP90-peptide complex can bepurified from 1 g of cells/tissue.

[0104] 4.3.3. Preparation and Purification of GP96-Peptide Complexes

[0105] A procedure that can be used, presented by way of example but notlimitation, is described below.

[0106] A pellet of tumors is resuspended in 3 volumes of bufferconsisting of 30 mM sodium bicarbonate buffer (pH 7.5) and 1 mM PMSF andthe cells allowed to swell on ice 20 minutes. The cell pellet is thenhomogenized in a Dounce homogenizer (the appropriate clearance of thehomogenizer will vary according to each cell type) on ice until >95%cells are lysed.

[0107] The lysate is centrifuged at 1,000 g for 10 minutes to removeunbroken cells, nuclei and other debris. The supernatant from thiscentrifugation step is then recentrifuged at 100,000 g for 90 minutes.The gp96-peptide complex can be purified either from the 100,000 pelletor from the supernatant.

[0108] When purified from the supernatant, the supernatant is dilutedwith equal volume of 2× lysis buffer and the supernatant mixed for 2-3hours at 4° C. with Con A Sepharose equilibrated with PBS containing 2mM Ca²⁺ and 2 mM Mg²⁺. Then, the slurry is packed into a column andwashed with 1× lysis buffer until the OD₂₈₀ drops to baseline. Then, thecolumn is washed with 1/3 column bed volume of 10% α-methyl mannoside(α-MM) dissolved in PBS containing 2 mM Ca²⁺ and 2 mM Mg²⁺, the columnsealed with a piece of parafilm, and incubated at 37° C. for 15 minutes.Then the column is cooled to room temperature and the parafilm removedfrom the bottom of the column. Five column volumes of the α-MM bufferare applied to the column and the eluate analyzed by SDS-PAGE. Typicallythe resulting material is about 60-95% pure, however this depends uponthe cell type and the tissue-to-lysis buffer ratio used. Then the sampleis applied to a Mono Q FPLC column (Pharmacia) equilibrated with abuffer containing 5 mM sodium phosphate, pH 7. The proteins are theneluted from the column with a 0-1M NaCl gradient and the gp96 fractionelutes between 400 mM and 550 mM NaCl.

[0109] The procedure, however, may be modified by two additional steps,used either alone or in combination, to consistently produce apparentlyhomogeneous gp96-peptide complexes. One optional step involves anammonium sulfate precipitation prior to the Con A purification step andthe other optional step involves DEAE-Sepharose purification after theCon A purification step but before the Mono Q FPLC step.

[0110] In the first optional step, described by way of example asfollows, the supernatant resulting from the 100,000 g centrifugationstep is brought to a final concentration of 50% ammonium sulfate by theaddition of ammonium sulfate. The ammonium sulfate is added slowly whilegently stirring the solution in a beaker placed in a tray of ice water.The solution is stirred from about ½ to 12 hours at 4° C. and theresulting solution centrifuged at 6,000 rpm (Sorvall SS34 rotor). Thesupernatant resulting from this step is removed, brought to 70% ammoniumsulfate saturation by the addition of ammonium sulfate solution, andcentrifuged at 6,000 rpm (Sorvall SS34 rotor). The resulting pellet fromthis step is harvested and suspended in PBS containing 70% ammoniumsulfate in order to rinse the pellet. This mixture is centrifuged at6,000 rpm (Sorvall SS34 rotor) and the pellet dissolved in PBScontaining 2 mM Ca²⁺ and Mg²⁺. Undissolved material is removed by abrief centrifugation at 15,000 rpm (Sorvall SS34 rotor). Then, thesolution is mixed with Con A Sepharose and the procedure followed asbefore.

[0111] In the second optional step, described by way of example asfollows, the gp96 containing fractions eluted from the Con A column arepooled and the buffer exchanged for 5 mM sodium phosphate buffer, pH 7,300 mM NaCl by dialysis, or preferably by buffer exchange on a SephadexG25 column. After buffer exchange, the solution is mixed withDEAE-Sepharose previously equilibrated with 5 mM sodium phosphatebuffer, pH 7, 300 mM NaCl. The protein solution and the beads are mixedgently for 1 hour and poured into a column. Then, the column is washedwith 5 mM sodium phosphate buffer, pH 7, 300 mM NaCl, until theabsorbance at 280 nm drops to baseline. Then, the bound protein iseluted from the column with five volumes of SmM sodium phosphate buffer,pH 7, 700 mM NaCl. Protein containing fractions are pooled and dilutedwith 5 mM sodium phosphate buffer, pH 7 in order to lower the saltconcentration to 175 mM. The resulting material then is applied to theMono Q FPLC column (Pharmacia) equilibrated with 5 mM sodium phosphatebuffer, pH 7 and the protein that binds to the Mono Q FPLC column(Pharmacia) is eluted as described before.

[0112] It is appreciated, however, that one skilled in the art mayassess, by routine experimentation, the benefit of incorporating thesecond optional step into the purification protocol. In addition, it isappreciated also that the benefit of adding each of the optional stepswill depend upon the source of the starting material.

[0113] When the gp96 fraction is isolated from the 100,000 g pellet, thepellet is suspended in 5 volumes of PBS containing either 1% sodiumdeoxycholate or 1% oxtyl glucopyranoside (but without the Mg²⁺ and Ca²⁺)and incubated on ice for 1 hour. The suspension is centrifuged at 20,000g for 30 minutes and the resulting supernatant dialyzed against severalchanges of PBS (also without the Mg²⁺ and Ca²⁺) to remove the detergent.The dialysate is centrifuged at 100,000 g for 90 minutes, thesupernatant harvested, and calcium and magnesium are added to thesupernatant to give final concentrations of 2 mM, respectively. Then thesample is purified by either the unmodified or the modified method forisolating gp96-peptide complex from the 1 00,000 g supernatant, seeabove.

[0114] The gp96-peptide complexes can be purified to apparenthomogeneity using this procedure. About 10-20 μg of gp96 can be isolatedfrom 1 g cells/tissue.

[0115] 4.3.4. Preparation and Purification of α2M

[0116] Alpha-2-macroglobulin can be bought from commercial sources orprepared by purifying it from human blood.

[0117] Generally, alpha-2-macroglobulin can be recovered and purifiedfrom sera of mammals by known methods, including ammonium sulfateprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, immunoaffinity chromatography,hydroxyapatite chromatography, and lectin chromatography.

[0118] In one embodiment, α2M are purified from serum using affinitypurification techniques. Methods for chromatography fractionation ofproteins, such as affinity chromatography, are well known in the art.Briefly, affinity chromatography utilizes an immobilized binding partnerto specifically capture the protein in the binding reaction. The bindingpartner molecule of the affinity capture assay can comprise, forexample, an antibody to α2M or other ligand, such as an α2M receptorbinding domain which specifically binds α2M. Alternatively, a filterbinding assay utilizes a device, such as a solid phase surface such as afilter or a column, to non-specifically retain proteins or proteincomplexes based on some physical or chemical difference between thecomplexes and the unbound reactants. Affinity chromatography and/orfilter binding separation techniques may be used to isolate α2M fromserum or other bodily fluid as described herein.

[0119] In a specific embodiment of the invention, α2M are isolated fromserum as follows: serum is contacted to a solid phase, such as anagarose column, which contains a binding partner of α2M, i.e., anα2M-binding molecule. The serum is allowed to incubate on the solidphase for a period of time sufficient to allow binding of α2M with thesolid phase. The material which does not bind is then removed from thesolid phase; and the bound α2M is eluted from the solid phase.

[0120] The binding partner of α2M may be any molecule which specificallybinds to α2M. In a preferred embodiment, the α2M-binding molecule is anantibody specific to α2M. The α2M-specific antibody is preferably amonoclonal antibody. In another preferred embodiment, the α2M-bindingmolecule is a ligand-binding fragment of the α2M receptor.

[0121] The solid phase may be any surface or matrix, such as, but notlimited to, polycarbonate, polystyrene, polypropylene, polyethylene,glass, nitrocellulose, dextran, nylon, polyacrylamide and agarose. Thesupport configuration can include beads, membranes, microparticles, theinterior surface of a reaction vessel such as a microtiter plate, testtube or other reaction vessel.

[0122] In a preferred embodiment, α2M are isolated from serum from miceby diluting serum 1:1 with 0.04 M Tris pH 7.6, 0.15 M NaCl. The mixtureis then applied to a 65 ml Sephacryl S 300R (Sigma) column equilibratedand eluted with the same buffer. α2M-positive fractions are determinedby dot blot and the buffer changed to a 0.01 M sodium phosphate bufferat pH 7.5 by use of a PD-10 column. Alternatively, the 0.04 M Tris pH7.6, 0.15 M NaCl buffer can be used as buffer in the 65 ml column toeliminate the step of exchanging the buffer. The complex-containingfractions are applied to a Concanavalin A sepharose column. Boundcomplex are eluted with 0.2M methylmannose pyranoside, or 5%methylmannose pyranoside, and applied to a DEAE column equilibrated with0.05M sodium acetate buffer. A2M are eluted in a pure form, as analyzedby SDS-PAGE and immunoblotting with 0.13 M sodium acetate buffer.

[0123] In yet another embodiment, α2M can be isolated from blood, thefollowing non-limiting protocol can be used by way of example: blood iscollected from a subject and is allowed to clot. It is then centrifugedfor 30 minutes under 14,000×g to obtain the serum which is then appliedto a gel filtration column (Sephacryl S-300R) equilibrated with 0.04MTris buffer pH 7.6 plus 0.3M NaCl. A 65 ml column is used for about 10ml of serum. Three ml fractions are collected and each fraction istested for the presence of α2M by dot blot using an α2M specificantibody. The α2M positive fractions are pooled and applied to a PD10column to exchange the buffer to 0.01M Sodium Phosphate buffer pH 7.5with PMSF. The pooled fractions are then applied to a Con A column (10ml) equilbrated with the phosphate buffer. The column is washed and theprotein is eluted with 5% methylmannose pyranoside. The eluent is passedover a PD10 column to change the buffer to a Sodium Acetate buffer(0.05M; pH6.0). A DEAE column is then equilibrated with acetate bufferand the sample is applied to the DEAE column. The column is washed andthe protein is eluted with 0.13M sodium acetate. The fractions with α2Mare then pooled. The α2M can be purified to apparent homogeneity usingthis procedure as assayed by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis.

[0124] Other methods for isolation of α2M known in the art can also beused (Dubin et al., 1984, Immunotherapy 8(4):589-596,; Okubo et al.,1981, Bio. Chem. Biophys. 688:257-267; Nieuwenhuizen et al. 1979,Biochem. Et Biophy. 580:129-139).

[0125] 4.3.5. Preparation and Purification of Noncovalent CellularlyProduced HSP110-Peptide Complexes

[0126] A procedure, described by Wang et al., 2001, J. Immunol.166(1):490-7, that can be used, presented by way of example and notlimitation, is as follows:

[0127] A pellet (40-60 ml) of cell or tissue, e.g., tumor cell tissue,is homogenized in 5 vol of hypotonic buffer (30 mN sodium bicarbonate,pH7.2, and protease inhibitors) by Dounce homogenization. The lysate iscentrifuged at 4,500×g and then 100,000×g for 2 hours. If the cells ortissues are of hepatic origin, the resulting supernatant is was firstapplied to a blue Sepharose column (Pharmacia) to remove albumin.Otherwise, the resulting supernatant is applied to a Con A-Sepharosecolumn (Pharmacia Biotech, Piscataway, N.J.) previously equilibratedwith binding buffer (20 mM Tris-HCl, pH 7.5; 100 mM NaCl; 1 mM MgCl₂; 1mM CaCl₂; 1 mM MnCl₂; and 15 mM 2-ME). The bound proteins are elutedwith binding buffer containing 15% α-D-o-methylmannoside (Sigma, St.Louis, Mo.).

[0128] Con A-Sepharose unbound material is first dialyzed against asolution of 20 mM Tris-HCl, pH 7.5; 100 mM NaCl; and 15 mM 2-ME, andthen applied to a DEAE-Sepharose column and eluted by salt gradient from100 to 500 mM NaCl. Fractions containing hsp 110 are collected,dialyzed, and loaded onto a Mono Q (Pharmacia) 10/10 column equilibratedwith 20 mM Tris-HCl, pH 7.5; 200 mM NaCl; and 15 mM 2-ME. The boundproteins are eluted with a 200-500 mM NaCl gradient. Fractions areanalyzed by SDS-PAGE followed by immunoblotting with an Ab for hsp 110,as described by Wang et al., 1999, J. Immunol. 162:3378. Pooledfractions containing hsp 110 are concentrated by Centriplus (Amicon,Beverly, Mass.) and applied to a Superose 12 column (Pharmacia).Proteins are eluted by 40 mM Tris-HCl, pH 8.0; 150 mM NaCl; and 15 mM2-ME with a flow rate of 0.2 ml/min.

[0129] 4.3.6. Preparation and Purification of Noncovalent CellularlyProduced GRP170-Peptide Complexes

[0130] A procedure, described by Wang et al., 2001, J. Immunol.166(1):490-7, that can be used, presented by way of example and notlimitation, is as follows:

[0131] A pellet (40-60 ml) of cell or tissue, e.g., tumor cell tissue,is homogenized in 5 vol of hypotonic buffer (30 mN sodium bicarbonate,pH7.2, and protease inhibitors) by Dounce homogenization. The lysate iscentrifuged at 4,500×g and then 100,000×g for 2 hours. If the cells ortissues are of hepatic origin, the resulting supernatant is was firstapplied to a blue Sepharose column (Pharmacia) to remove albumin.Otherwise, the resulting supernatant is applied to a Con A-Sepharosecolumn (Pharmacia Biotech, Piscataway, N.J.) previously equilibratedwith binding buffer (20 mM Tris-HCl, pH 7.5; 100 mM NaCl; 1 mM MgCl₂; 1mM CaCl₂; 1 mM MnCl₂; and 15 mM 2-ME). The bound proteins are elutedwith binding buffer containing 15% α-D-o-methylmannoside (Sigma, St.Louis, Mo.).

[0132] Con A-Sepharose-bound material is first dialyzed against 20 mMTris-HCl, pH 7.5, and 150 mM NaCl and then applied to a Mono Q columnand eluted by a 150 to 400 mM NaCl gradient. Pooled fractions areconcentrated and applied on the Superose 12 column (Pharmacia).Fractions containing homogeneous grp 170 are collected.

[0133] 4.3.7. Recombinant Expression of Heat Shock Proteins and α2M

[0134] In certain embodiments of the present invention, HSPs and α2M canbe prepared from cells that express higher levels of HSPs and α2Mthrough recombinant means. Amino acid sequences and nucleotide sequencesof many HSPs and α2M are generally available in sequence databases, suchas GenBank. Computer programs, such as Entrez, can be used to browse thedatabase, and retrieve any amino acid sequence and genetic sequence dataof interest by accession number. These databases can also be searched toidentify sequences with various degrees of similarities to a querysequence using programs, such as FASTA and BLAST, which rank the similarsequences by alignment scores and statistics. Such nucleotide sequencesof non-limiting examples of HSPs that can be used for the compositions,methods, and for preparation of the HSP peptide-complexes of theinvention are as follows: human HSP70, Genbank Accession No. M24743,Hunt et al., 1995, Proc. Natl. Acad. Sci. U.S.A., 82: 6455-6489; humanHSP90, Genbank Accession No. X15183, Yamazaki et al., Nucl. Acids Res.17: 7108; human gp96: Genbank Accession No. X15187, Maki et al., 1990,Proc. Natl. Acad. Sci. U.S.A. 87: 5658-5562; human BiP: GenbankAccession No. M19645; Ting et al., 1988, DNA 7: 275-286; human HSP27,Genbank Accession No. M24743; Hickey et al., 1986, Nucleic Acids Res.14: 4127-45; mouse HSP70: Genbank Accession No. M35021, Hunt et al.,1990, Gene 87: 199-204; mouse gp96: Genbank Accession No. M16370,Srivastava et al., 1987, Proc. Natl. Acad. Sci. U.S.A. 85: 3807-3811;and mouse BiP: Genbank Accession No. U16277, Haas et al., 1988, Proc.Natl. Acad. Sci. U.S.A. 85: 2250-2254. Degenerate sequences encodingHSPs can also be used.

[0135] As used herein, the term “α2M” embraces other polypeptidefragments, analogs, and variants of α2M having at least 35% to 55%,preferably 55% to 75%, and most preferably 75% to 85% amino acididentity with α2M, and is capable of forming a complex with an antigenicpeptide, which complex is capable of being taken up by an antigenpresenting cell and eliciting an immune response against the antigenicmolecule. The α2M molecule of the invention can be purchasedcommercially or purified from natural sources (Kurecki et al., 1979,Anal. Biochem. 99:415-420), chemically synthesized, or recombinantlyproduced. Non-limiting examples of α2M sequences that can be used forpreparation of the α2M polypeptides of the invention are as follows:Genbank Accession Nos. M11313, P01023, AAA51551; Kan et al., 1985, Proc.Nat. Acad. Sci. 82: 2282-2286. A degenerate sequence encoding α2M canalso be used.

[0136] Once the nucleotide sequence encoding the HSP or α2M of choicehas been identified, the nucleotide sequence, or a fragment thereof, canbe obtained and cloned into an expression vector for recombinantexpression. The expression vector can then be introduced into a hostcell for propagation of the HSP or α2M. Methods for recombinantproduction of HSPs or α2M are described in detail herein.

[0137] The DNA may be obtained by DNA amplification or molecular cloningdirectly from a tissue, cell culture, or cloned DNA (e.g., a DNA“library”) using standard molecular biology techniques (see e.g.,Methods in Enzymology, 1987, volume 154, Academic Press; Sambrook et al.1989, Molecular Cloning—A Laboratory Manual, 2nd Edition, Cold SpringHarbor Press, New York; and Current Protocols in Molecular Biology,Ausubel et al. (eds.), Greene Publishing Associates and WileyInterscience, New York, each of which is incorporated herein byreference in its entirety). Clones derived from genomic DNA may containregulatory and intron DNA regions in addition to coding regions; clonesderived from cDNA will contain only exon sequences. Whatever the source,the HSP or α2M gene should be cloned into a suitable vector forpropagation of the gene.

[0138] In a preferred embodiment, DNA can be amplified from genomic orcDNA by polymerase chain reaction (PCR) amplification using primersdesigned from the known sequence of a related or homologous HSP or α2M.PCR is used to amplify the desired sequence in DNA clone or a genomic orcDNA library, prior to selection. PCR can be carried out, e.g., by useof a thermal cycler and Taq polymerase (Gene Amp®). The polymerase chainreaction (PCR) is commonly used for obtaining genes or gene fragments ofinterest. For example, a nucleotide sequence encoding an HSP or α2M ofany desired length can be generated using PCR primers that flank thenucleotide sequence encoding open reading fram. Alternatively, an HSP orα2M gene sequence can be cleaved at appropriate sites with restrictionendonuclease(s) if such sites are available, releasing a fragment of DNAencoding the HSP or α2M gene. If convenient restriction sites are notavailable, they may be created in the appropriate positions bysite-directed mutagenesis and/or DNA amplification methods known in theart (see, for example, Shankarappa et al., 1992, PCR Method Appl. 1:277-278). The DNA fragment that encodes the HSP or α2M is then isolated,and ligated into an appropriate expression vector, care being taken toensure that the proper translation reading frame is maintained.

[0139] In an alternative embodiment, for the molecular cloning of an HSPor α2M gene from genomic DNA, DNA fragments are generated to form agenomic library. Since some of the sequences encoding related HSPs orα2M are available and can be purified and labeled, the cloned DNAfragments in the genomic DNA library may be screened by nucleic acidhybridization to a labeled probe (Benton and Davis, 1977, Science 196:180; Grunstein and Hogness, 1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961). Those DNA fragments with substantial homology to the probe willhybridize. It is also possible to identify an appropriate fragment byrestriction enzyme digestion(s) and comparison of fragment sizes withthose expected according to a known restriction map.

[0140] Alternatives to isolating the HSP or α2M genomic DNA include, butare not limited to, chemically synthesizing the gene sequence itselffrom a known sequence or synthesizing a cDNA to the mRNA which encodesthe HSP or α2M. For example, RNA for cDNA cloning of the HSP or α2M genecan be isolated from cells which express the HSP or α2M. A cDNA librarymay be generated by methods known in the art and screened by methods,such as those disclosed for screening a genomic DNA library. If anantibody to the HSP or α2M is available, the HSP or α2M may beidentified by binding of a labeled antibody to the HSP- orα2M-synthesizing clones.

[0141] Other specific embodiments for the cloning of a nucleotidesequence encoding an HSP or α2M, are presented as examples but not byway of limitation, as follows: In a specific embodiment, nucleotidesequences encoding an HSP or α2M can be identified and obtained byhybridization with a probe comprising a nucleotide sequence encoding HSPor α2M under various conditions of stringency which are well known inthe art (including those employed for cross-species hybridizations).

[0142] Any technique for mutagenesis known in the art can be used tomodify individual nucleotides in a DNA sequence, for purpose of makingamino acid substitution(s) in the expressed peptide sequence, or forcreating/deleting restriction sites to facilitate further manipulations.Such techniques include but are not limited to, chemical mutagenesis, invitro site-directed mutagenesis (Hutchinson et al., 1978, J. Biol. Chem.253: 6551), oligonucleotide-directed mutagenesis (Smith, 1985, Ann. Rev.Genet. 19: 423-463; Hill et al., 1987, Methods Enzymol. 155: 558-568),PCR-based overlap extension (Ho et al., 1989, Gene 77: 51-59), PCR-basedmegaprimer mutagenesis (Sarkar et al., 1990, Biotechniques 8: 404-407),etc. Modifications can be confirmed by double stranded dideoxynucleotideDNA sequencing.

[0143] In certain embodiments, a nucleic acid encoding a secretory formof a non-secreted HSP is used to practice the methods of the presentinvention. Such a nucleic acid can be constructed by deleting the codingsequence for the ER retention signal, KDEL. Optionally, the KDEL codingsequence is replaced with a molecular tag to facilitate the recognitionand purification of the HSP, such as the Fc portion of murine IgG1. Inanother embodiment, a molecular tag can be added to naturally secretedHSPs or α2M. PCT publication no. WO 99/42121 demonstrates that deletionof the ER retention signal of gp96 resulted in the secretion of gp96-Igpeptide-complexes from transfected tumor cells, and the fusion of theKDEL-deleted gp96 with murine IgG1 facilitated its detection by ELISAand FACS analysis and its purification by affinity chromatography withthe aid of Protein A.

[0144] 4.3.7.1 Expression Systems

[0145] Nucleotide sequences encoding an HSP or α2M molecule can beinserted into the expression vector for propagation and expression inrecombinant cells. An expression construct, as used herein, refers to anucleotide sequence encoding an HSP or α2M operably associated with oneor more regulatory regions which allows expression of the HSP or α2Mmolecule in an appropriate host cell. “Operably-associated” refers to anassociation in which the regulatory regions and the HSP or α2Mpolypeptide sequence to be expressed are joined and positioned in such away as to permit transcription, and ultimately, translation of the HSPor α2M sequence. A variety of expression vectors may be used for theexpression of HSPs or α2M, including, but not limited to, plasmids,cosmids, phage, phagemids, or modified viruses. Examples includebacteriophages such as lambda derivatives, or plasmids such as pBR322 orpUC plasmid derivatives or the Bluescript vector (Stratagene).Typically, such expression vectors comprise a functional origin ofreplication for propagation of the vector in an appropriate host cell,one or more restriction endonuclease sites for insertion of the HSP orα2M gene sequence, and one or more selection markers.

[0146] For expression of HSPs or α2M in mammalian host cells, a varietyof regulatory regions can be used, for example, the SV40 early and latepromoters, the cytomegalovirus (CMV) immediate early promoter, and theRous sarcoma virus long terminal repeat (RSV-LTR) promoter. Induciblepromoters that may be useful in mammalian cells include but are notlimited to those associated with the metallothionein II gene, mousemammary tumor virus glucocorticoid responsive long terminal repeats(MMTV-LTR), the β-interferon gene, and the HSP70 gene (Williams et al.,1989, Cancer Res. 49: 2735-42; Taylor et al., 1990, Mol. Cell. Biol. 10:165-75). The efficiency of expression of the HSP or α2M in a host cellmay be enhanced by the inclusion of appropriate transcription enhancerelements in the expression vector, such as those found in SV40 virus,Hepatitis B virus, cytomegalovirus, immunoglobulin genes,metallothionein, β-actin (see Bittner et al., 1987, Methods in Enzymol.153: 516-544; Gorman, 1990, Curr. Op. in Biotechnol. 1: 36-47).

[0147] The expression vector may also contain sequences that permitmaintenance and replication of the vector in more than one type of hostcell, or integration of the vector into the host chromosome. Suchsequences may include but are not limited to replication origins,autonomously replicating sequences (ARS), centromere DNA, and telomereDNA. It may also be advantageous to use shuttle vectors that can bereplicated and maintained in at least two types of host cells.

[0148] In addition, the expression vector may contain selectable orscreenable marker genes for initially isolating or identifying hostcells that contain DNA encoding an HSP or α2M. For long term, high yieldproduction of HSPs or α2M, stable expression in mammalian cells ispreferred. A number of selection systems may be used for mammaliancells, including, but not limited, to the Herpes simplex virus thymidinekinase (Wigler et al., 1977, Cell 11: 223), hypoxanthine-guaninephosphoribosyltransferase (Szybalski and Szybalski, 1962, Proc. Natl.Acad. Sci. U.S.A. 48: 2026), and adenine phosphoribosyltransferase (Lowyet al., 1980, Cell 22: 817) genes can be employed in tk⁻, hgprt⁻ oraprt⁻ cells, respectively. Also, antimetabolite resistance can be usedas the basis of selection for dihydrofolate reductase (dhfr), whichconfers resistance to methotrexate (Wigler et al., 1980, Natl. Acad.Sci. U.S.A. 77: 3567; O'Hare et al., 1981, Proc. Natl. Acad. Sci. U.S.A.78: 1527); gpt, which confers resistance to mycophenolic acid (Mulliganand Berg, 1981, Proc. Natl. Acad. Sci. U.S.A. 78: 2072); neomycinphosphotransferase (neo), which confers resistance to the aminoglycosideG-418 (Colberre-Garapin et al., 1981, J. Mol. Biol. 150: 1); andhygromycin phosphotransferase (hyg), which confers resistance tohygromycin (Santerre et al., 1984, Gene 30: 147). Other selectablemarkers, such as but not limited to histidinol and Zeocin™ can also beused.

[0149] The expression construct comprising an HSP- or α2M-codingsequence operably associated with regulatory regions can be directlyintroduced into appropriate host cells for expression and production ofthe HSP or α2M complexes of the invention without further cloning (see,for example, U.S. Pat. No. 5,580,859). The expression constructs mayalso contain DNA sequences that facilitate integration of the codingsequence into the genome of the host cell, e.g., via homologousrecombination. In this instance, it is not necessary to employ anexpression vector comprising a replication origin suitable forappropriate host cells in order to propagate and express the HSP or α2Mmolecule in the host cells.

[0150] Expression constructs containing cloned HSP or α2M codingsequences can be introduced into the mammalian host cell by a variety oftechniques known in the art, including but not limited to calciumphosphate mediated transfection (Wigler et al., 1977, Cell 11: 223-232),liposome-mediated transfection (Schaefer-Ridder et al., 1982, Science215: 166-168), electroporation (Wolff et al., 1987, Proc. Natl. Acad.Sci. 84: 3344), and microinjection (Cappechi, 1980, Cell 22: 479-488).

[0151] Any of the cloning and expression vectors described herein may besynthesized and assembled from known DNA sequences by techniques wellknown in the art. The regulatory regions and enhancer elements can be ofa variety of origins, both natural and synthetic. Some vectors and hostcells may be obtained commercially. Non-limiting examples of usefulvectors are described in Appendix 5 of Current Protocols in MolecularBiology, 1988, ed. Ausubel et al., Greene Publish. Assoc. & WileyInterscience, which is incorporated herein by reference; and thecatalogs of commercial suppliers such as Clontech Laboratories,Stratagene Inc., and Invitrogen, Inc.

[0152] Alternatively, number of viral-based expression systems may alsobe utilized with mammalian cells for recombinant expression of HSPs orα2M. Vectors using DNA virus backbones have been derived from simianvirus 40 (SV40) (Hamer et al., 1979, Cell 17: 725), adenovirus (VanDoren et al., 1984, Mol. Cell Biol. 4: 1653), adeno-associated virus(McLaughlin et al., 1988, J. Virol. 62: 1963), and bovine papillomasvirus (Zinn et al., 1982, Proc. Natl. Acad. Sci. 79: 4897). In caseswhere an adenovirus is used as an expression vector, the donor DNAsequence may be ligated to an adenovirus transcription/translationcontrol region, e.g., the late promoter and tripartite leader sequence.This chimeric gene may then be inserted in the adenovirus genome by invitro or in vivo recombination. Insertion in a non-essential region ofthe viral genome (e.g., region E1 or E3) will result in a recombinantvirus that is viable and capable of expressing heterologous products ininfected hosts (see, e.g., Logan and Shenk, 1984, Proc. Natl. Acad. Sci.U.S.A. 81: 3655-3659).

[0153] Bovine papillomavirus (BPV) can infect many higher vertebrates,including man, and its DNA replicates as an episome. A number of shuttlevectors have been developed for recombinant gene expression which existas stable, multicopy (20-300 copies/cell) extrachromosomal elements inmammalian cells. Typically, these vectors contain a segment of BPV DNA(the entire genome or a 69% transforming fragment), a promoter with abroad host range, a polyadenylation signal, splice signals, a selectablemarker, and “poisonless” plasmid sequences that allow the vector to bepropagated in E. coli. Following construction and amplification inbacteria, the expression gene construct is transfected into culturedmammalian cells, for example, by the techniques of calcium phosphatecoprecipitation or electroporation. For those host cells that do notmanifest a transformed phenotype, selection of transformants is achievedby use of a dominant selectable marker, such as histidinol and G418resistance. For example, BPV vectors such as pBCMGSNeo and pBCMGHis maybe used to express HSPs or α2M (Karasuyama et al., Eur. J. Immunol. 18:97-104; Ohe et al., Human Gene Therapy 6: 325-33) which may then betransfected into a diverse range of cell types for HSP or α2Mexpression.

[0154] Alternatively, the vaccinia 7.5K promoter may be used (see, e.g.,Mackett et al., 1982, Proc. Natl. Acad. Sci. U.S.A. 79: 7415-7419;Mackett et al., 1984, J. Virol. 49: 857-864; Panicali et al., 1982,Proc. Natl. Acad. Sci. U.S.A. 79: 4927-4931) In cases where a human hostcell is used, vectors based on the Epstein-Barr virus (EBV) origin(OriP) and EBV nuclear antigen 1 (EBNA-1; a trans-acting replicationfactor) may be used. Such vectors can be used with a broad range ofhuman host cells, e.g., EBO-pCD (Spickofsky et al., 1990, DNA Prot. Eng.Tech. 2: 14-18), pDR2 and λDR2 (available from Clontech Laboratories).

[0155] Recombinant HSP or α2M expression can also be achieved by aretrovirus-based expression system. In contrast to transfection,retroviruses can efficiently infect and transfer genes to a wide rangeof cell types including, for example, primary hematopoietic cells. Inretroviruses such as Moloney murine leukemia virus, most of the viralgene sequences can be removed and replaced with an HSP or α2M codingsequence, while the missing viral functions can be supplied in trans.The host range for infection by a retroviral vector can also bemanipulated by the choice of envelope used for vector packaging.

[0156] For example, a retroviral vector can comprise a 5′ long terminalrepeat (LTR), a 3′ LTR, a packaging signal, a bacterial origin ofreplication, and a selectable marker. The ND-associated antigenicpeptide DNA is inserted into a position between the 5′ LTR and 3′ LTR,such that transcription from the 5′ LTR promoter transcribes the clonedDNA. The 5′ LTR comprises a promoter, including but not limited to anLTR promoter, an R region, a U5 region and a primer binding site, inthat order. Nucleotide sequences of these LTR elements are well known inthe art. A heterologous promoter as well as multiple drug selectionmarkers may also be included in the expression vector to facilitateselection of infected cells (see McLauchlin et al., 1990, Prog. NucleicAcid Res. and Molec. Biol. 38: 91-135; Morgenstern et al., 1990, NucleicAcid Res. 18: 3587-3596; Choulika et al., 1996, J. Virol 70: 1792-1798;Boesen et al., 1994, Biotherapy 6: 291-302; Salmons and Gunzberg, 1993,Human Gene Therapy 4: 129-141; and Grossman and Wilson, 1993, Curr.Opin. in Genetics and Devel. 3: 110-114).

[0157] The recombinant cells may be cultured under standard conditionsof temperature, incubation time, optical density, and media composition.Alternatively, cells may be cultured under conditions emulating thenutritional and physiological requirements of a cell in which the HSP isendogenously expressed. Modified culture conditions and media may beused to enhance production of HSP-peptide complexes. For example,recombinant cells may be grown under conditions that promote inducibleHSP expression.

[0158] Alpha-2-macroglobulin and HSP polypeptides of the invention maybe expressed as fusion proteins to facilitate recovery and purificationfrom the cells in which they are expressed. For example, an HSP or α2Mpolypeptide may contain a signal sequence leader peptide to direct itstranslocation across the ER membrane for secretion into culture medium.Further, an HSP or α2M polypeptide may contain an affinity label, suchas a affinity label, fused to any portion of the HSP or α2M polypeptidenot involved in binding antigenic peptide, such as for example, thecarboxyl terminal. The affinity label can be used to facilitatepurification of the protein, by binding to an affinity partner molecule.

[0159] Various methods for production of such fusion proteins are wellknown in the art. The manipulations which result in their production canoccur at the gene or protein level, preferably at the gene level. Forexample, the cloned coding region of an HSP or α2M polypeptide may bemodified by any of numerous recombinant DNA methods known in the art(Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Ausubel et al.,in Chapter 8 of Current Protocols in Molecular Biology, GreenePublishing Associates and Wiley Interscience, New York). It will beapparent from the following discussion that substitutions, deletions,insertions, or any combination thereof are introduced or combined toarrive at a final nucleotide sequence encoding an HSP or α2Mpolypeptide.

[0160] In various embodiments, fusion proteins comprising the HSP or α2Mpolypeptide may be made using recombinant DNA techniques. For example, arecombinant gene encoding an HSP or α2M polypeptide may be constructedby introducing an HSP or α2M gene fragment in the proper reading frameinto a vector containing the sequence of an affinity label, such thatthe HSP or α2M polypeptide is expressed as a peptide-tagged fusionprotein. Affinity labels, which may be recognized by specific bindingpartners, may be used for affinity purification of the HSP or α2Mpolypeptide.

[0161] In a preferred embodiment, the affinity label is fused at itsamino terminal to the carboxyl terminal of HSP or α2M. The precise siteat which the fusion is made in the carboxyl terminal is not critical.The optimal site can be determined by routine experimentation.

[0162] A variety of affinity labels known in the art may be used, suchas, but not limited to, the immunoglobulin constant regions,polyhistidine sequence (Petty, 1996, Metal-chelate affinitychromatography, in Current Protocols in Molecular Biology, Vol. 2, Ed.Ausubel et al., Greene Publish. Assoc. & Wiley Interscience),glutathione S-transferase (GST; Smith, 1993, Methods Mol. Cell Bio.4:220-229), the E. coli maltose binding protein (Guan et al., 1987, Gene67:21-30), and various cellulose binding domains (U.S. Pat. Nos.5,496,934; 5,202,247; 5,137,819; Tomme et al., 1994, Protein Eng.7:117-123), etc. Other affinity labels may impart fluorescent propertiesto an HSP or α2M polypeptide, e.g., portions of green fluorescentprotein and the like. Other possible affinity labels are short aminoacid sequences to which monoclonal antibodies are available, such as butnot limited to the following well known examples, the FLAG epitope, themyc epitope at amino acids 408-439, the influenza virus hemagglutinin(HA) epitope. Other affinity labels are recognized by specific bindingpartners and thus facilitate isolation by affinity binding to thebinding partner which can be immobilized onto a solid support. Someaffinity labels may afford the HSP or α2M polypeptide novel structuralproperties, such as the ability to form multimers. Dimerization of anHSP or α2M polypeptide with a bound peptide may increase avidity ofinteraction between the HSP or α2M polypeptide and its partner in thecourse of antigen presentation. These affinity labels are usuallyderived from proteins that normally exist as homopolymers. Affinitylabels such as the extracellular domains of CD8 (Shiue et al., 1988, J.Exp. Med. 168:1993-2005), or CD28 (Lee et al., 1990, J. Immunol.145:344-352), or portions of the immunoglobulin molecule containingsites for interchain disulfide bonds, could lead to the formation ofmultimers. As will be appreciated by those skilled in the art, manymethods can be used to obtain the coding region of the above-mentionedaffinity labels, including but not limited to, DNA cloning, DNAamplification, and synthetic methods. Some of the affinity labels andreagents for their detection and isolation are available commercially.

[0163] A preferred affinity label is a non-variable portion of theimmunoglobulin molecule. Typically, such portions comprise at least afunctionally operative CH2 and CH3 domain of the constant region of animmunoglobulin heavy chain. Fusions are also made using the carboxylterminus of the Fc portion of a constant domain, or a region immediatelyamino-terminal to the CH1 of the heavy or light chain. Suitableimmunoglobulin-based affinity label may be obtained from IgG-1, -2, -3,or -4 subtypes, IgA, IgE, IgD, or IgM, but preferably IgG1. Preferably,a human immunoglobulin is used when the HSP or α2M polypeptide isintended for in vivo use for humans. Many DNA encoding immunoglobulinlight or heavy chain constant regions is known or readily available fromcDNA libraries. See, for example, Adams et al., Biochemistry, 1980,19:2711-2719; Gough et al., 1980, Biochemistry, 19:2702-2710; Dolby etal., 1980, Proc. Natl. Acad. Sci. U.S.A., 77:6027-6031; Rice et al.,1982, Proc. Natl. Acad. Sci. U.S.A., 79:7862-7865; Falkner et al., 1982,Nature, 298:286-288; and Morrison et al., 1984, Ann. Rev. Immunol,2:239-256. Because many immunological reagents and labeling systems areavailable for the detection of immunoglobulins, the HSP or α2Mpolypeptide—Ig fusion protein can readily be detected and quantified bya variety of immunological techniques known in the art, such as the useof enzyme-linked immunosorbent assay (ELISA), immunoprecipitation,fluorescence activated cell sorting (FACS), etc. Similarly, if theaffinity label is an epitope with readily available antibodies, suchreagents can be used with the techniques mentioned above to detect,quantitate, and isolate the HSP or α2M polypeptide containing theaffinity label. In many instances, there is no need to develop specificantibodies to the HSP or α2M polypeptide.

[0164] A particularly preferred embodiment is a fusion of an HSP or α2Mpolypeptide to the hinge, the CH2 and CH3 domains of humanimmunoglobulin G-1 (IgG-1; see Bowen et al., 1996, J. Immunol.156:442-49). This hinge region contains three cysteine residues whichare normally involved in disulfide bonding with other cysteines in theIg molecule. Since none of the cysteines are required for the peptide tofunction as a tag, one or more of these cysteine residues may optionallybe substituted by another amino acid residue, such as for example,serine.

[0165] Various leader sequences known in the art can be used for theefficient secretion of HSP or α2M polypeptide from bacterial andmammalian cells (von Heijne, 1985, J. Mol. Biol. 184:99-105). Leaderpeptides are selected based on the intended host cell, and may includebacterial, yeast, viral, animal, and mammalian sequences. For example,the herpes virus glycoprotein D leader peptide is suitable for use in avariety of mammalian cells. A preferred leader peptide for use inmammalian cells can be obtained from the V-J2-C region of the mouseimmunoglobulin kappa chain (Bernard et al., 1981, Proc. Natl. Acad. Sci.78:5812-5816). Preferred leader sequences for targeting HSP or α2Mpolypeptide expression in bacterial cells include, but are not limitedto, the leader sequences of the E. coli proteins OmpA (Hobom et al.,1995, Dev. Biol. Stand. 84:255-262), Pho A (Oka et al., 1985, Proc.Natl. Acad. Sci 82:7212-16), OmpT (Johnson et al., 1996, ProteinExpression 7:104-113), LamB and OmpF (Hoffman & Wright, 1985, Proc.Natl. Acad. Sci. USA 82:5107-5111), β-lactamase (Kadonaga et al., 1984,J. Biol. Chem. 259:2149-54), enterotoxins (Morioka-Fujimoto et al.,1991, J. Biol. Chem. 266:1728-32), and the Staphylococcus aureus proteinA (Abrahmsen et al., 1986, Nucleic Acids Res. 14:7487-7500), and the B.subtilis endoglucanase (Lo et al., Appl. Environ. Microbiol.54:2287-2292), as well as artificial and synthetic signal sequences(MacIntyre et al., 1990, Mol. Gen. Genet. 221:466-74; Kaiser et al.,1987, Science, 235:312-317).

[0166] DNA sequences encoding a desired affinity label or leaderpeptide, which may be readily obtained from libraries, producedsynthetically, or may be available from commercial suppliers, aresuitable for the practice of this invention. Such methods are well knownin the art.

4.4. Complexing Proteins and Peptides to HSP and α2M

[0167] Described herein are exemplary methods for complexing in vitrothe HSP or α2M with a population of proteins and/or peptides which havebeen prepared from antigenic cells, a cellular fraction thereof, orviral particles. The population of proteins and/or peptides are from aprotein preparation of the antigenic cells as described in Section4.2.1. In certain embodiments, the peptides are the result of digestionof a protein preparation of antigenic cells, a cellular fractionthereof, or viral particles. The complexing reaction can result in theformation of a covalent bond between a HSP and a protein or peptide ofthe antigenic cell or viral particle. The complexing reaction can resultin the formation of a covalent bond between a α2M and a protein orpeptide of the antigenic cell or viral particle. The complexing reactioncan also result in the formation of a non-covalent association between aHSP and a protein and/or a peptide, or a α2M and a protein and/or apeptide.

[0168] Prior to complexing, the HSPs can be pretreated with ATP orexposed to acidic conditions to remove any peptides that may benon-covalently associated with the HSP of interest. When the ATPprocedure is used, excess ATP is removed from the preparation by theaddition of apyranase as described by Levy, et al., 1991, Cell67:265-274. When acidic conditions are used, the buffer is readjusted toneutral pH by the addition of pH modifying reagents. A preferred,exemplary protocol for the noncovalent complexing of a population ofpeptides (average length between 7 to 20 amino acids) to an HSP in vitrois discussed below:

[0169] The population of peptides (1 μg, which can be dissolved in 10%to 50% dimethyl sulfoxide) and the pretreated HSP (9 μg) are admixed togive an approximately 5 peptides (or proteins): 1 HSP molar ratio. Then,the mixture is incubated for 15 minutes to 3 hours at 4° to 45° C. in asuitable binding buffer such as phosphate buffered saline pH7.4, or onecontaining 20 mM sodium phosphate, pH 7.2, 350 mM NaCl, 3 mM MgCl₂ and 1mM phenyl methyl sulfonyl fluoride (PMSF). The preparations arecentrifuged through a Centricon 10 assembly (Millipore) to remove anyunbound peptide. The non-covalent association of the proteins/peptideswith the HSPs can be assayed by High Performance Liquid Chromatography(HPLC) or Mass Spectrometry (MS).

[0170] In an alternative embodiment of the invention, preferred forproducing non-covalent complexes of HSP70 to proteins/peptides, 5-10micrograms of purified HSP70 is incubated with equimolar quantities ofproteins/peptides in 20 mM sodium phosphate buffer pH 7.5, 0.5M NaCl, 3mM MgCl₂ and 1 mM ADP in a volume of 100 microliter at 37° C. for 1 hr.This incubation mixture is centrifuged one or more times if necessary,through a Centricon 10 assembly (Millipore) to remove any unboundpeptide.

[0171] In an alternative embodiment of the invention, preferred forproducing non-covalent complexes of gp96 or HSP90 to peptides, 5-10micrograms of purified gp96 or HSP90 is incubated with equimolar orexcess quantities of the proteins/peptides in a suitable buffer such asone containing 20 mM sodium phosphate buffer pH 7.5, 0.5M NaCl, 3 mMMgCl2 at 60-65° C. for 5-20 min. This incubation mixture is allowed tocool to room temperature and centrifuged one or more times if necessary,through a Centricon 10 assembly (Millipore) to remove any unboundpeptide.

[0172] Following complexing with antigenic proteins and/or antigenicpeptides, an immunogenic HSP complex or α2M complex can optionally beassayed using, for example, the mixed lymphocyte target cell assay(MLTC) described below. Once HSP-peptide complexes and/or HSP-proteincomplexes have been isolated and diluted, they can be optionallycharacterized further in animal models using the preferredadministration protocols and excipients discussed below.

[0173] As an alternative to making non-covalent complexes of HSPs andproteins/peptides, a population of proteins/peptides can be covalentlyattached to HSPs.

[0174] In one embodiment, HSPs are covalently coupled to proteins and/orpeptides in a protein preparation by chemical crosslinking. Chemicalcrosslinking methods are well known in the art. For example, in apreferred embodiment, glutaraldehyde crosslinking may be used.Glutaradehyde crosslinking has been used for formation of covalentcomplexes of peptides and HSPs (see Barrios et al., 1992, Eur. J.Immunol. 22: 1365-1372). Preferably, 1-2 mg of HSP-peptide complex iscrosslinked in the presence of 0.002% glutaraldehyde for 2 hours.Glutaraldehyde is removed by dialysis against phosphate buffered saline(PBS) overnight (Lussow et al., 1991, Eur. J. Immunol. 21: 2297-2302).Alternatively, a HSP and a population of protein/peptides can becrosslinked by ultraviolet (UV) crosslinking under conditions known inthe art.

[0175] In another embodiment of the invention, a population of proteinsand/or peptides in a protein preparation can be non-covalently complexedto α2M by incubating the proteins/peptides with α2M at a 50:1 molarratio and incubated at 50° C. for 10 minutes followed by a 30 minuteincubation at 25° C. Free (uncomplexed) peptides can be removed by sizeexclusion filters. Complexes are preferably measured by a scintillationcounter to make sure that on a per molar basis, each HSP or α2M isobserved to bind equivalent amounts of proteins/peptide (approximately0.1% of the starting amount of the peptide). For details, see Binder,2001, J. Immunol. 166(8):4968-72, which is incorporated herein byreference in its entirety. To reduce the propensity of forming covalentcomplexes of α2M and the proteins and peptides in these reactions, itwill be desirable to inhibit or remove protease activity prior tocomplexing. This can be accomplished with the use of proteaseinhibitors, for example, by the methods described in section 4.2.1. Alsodesirable is adding a reducing agent (such as 2-mercaptoethanol) to thereactions to neutralize nucleophilic compounds present in the proteinpreparation which may activate α2M for covalent association.

[0176] In yet another embodiment, a population of antigenic proteinsand/or antigenic peptides in a protein preparation can be complexed toα2M covalently by methods as described in PCT publications WO 94/14976and WO 99/50303 for complexing a peptide to α2M, which are incorporatedherein by reference in their entirety. For example, antigenic proteinsand/or antigenic peptides can be incorporated into α2M by ammonia ormethylamine (or other small amine nucleophiles such as ethylamine)during reversal of the nucleophilic activation, employing heat (Grn andPizzo, 1998, Biochemistry, 37: 6009-6014; which is incorporated hereinby reference in its entirety). Such conditions that allow fortuitoustrapping of peptides by α2M can be employed to prepare the α2M complexesof the invention. Covalent linking of a population of antigenicproteins/peptides to α2M can also be performed using a bifunctionalcrosslinking agent. Such crosslinking agents and methods of their useare also well known in the art. Preferably, the crosslinking agent isinactivated and/or removed after the complexes are formed. Methods forcovalent coupling have been described previously (Osada et al., 1987,Biochem. Biophys. Res. Commun.146:26-31; Osada et al., 1988, Biochem.Biophys. Res. Commun. 150:883; Chu and Pizzo, 1993, J. Immunol. 150:48;Chu et al., 1994, Ann. N.Y. Acad. Sci. 737:291-307; Mitsuda et al.,1993, Biochem. Biophys. Res. Commun. 101:1326-1331).

[0177] In yet another embodiment, a population of proteins/peptides canbe complexed to a mixture of HSP and α2M in the same reaction by thenon-covalent or covalent methods described above.

[0178] Complexes of HSP and antigenic proteins and/or peptides fromseparate covalent and/or non-covalent complexing reactions canoptionally be combined to form a composition before administration to asubject. Complexes of α2M and antigenic proteins and/or peptides fromseparate covalent and/or non-covalent complexing reactions can alsooptionally be combined to form a composition before administration to asubject.

4.5. Prevention and Treatment of Cancer and Infectious Diseases

[0179] In accordance with the invention, a composition of the invention,which comprises complexes of antigenic peptides derived from digestedcytosolic and/or membrane-derived proteins of antigenic cells or viralparticle and a HSP and/or α2M, is administered to a subject with canceror an infectious disease. In one embodiment, “treatment” or “treating”refers to an amelioration of cancer or an infectious disease, or atleast one discernible symptom thereof. In another embodiment,“treatment” or “treating” refers to an amelioration of at least onemeasurable physical parameter associated with cancer or an infectiousdisease, not necessarily discernible by the subject. In yet anotherembodiment, “treatment” or “treating” refers to inhibiting theprogression of a cancer or an infectious disease, either physically,e.g., stabilization of a discernible symptom, physiologically, e.g.,stabilization of a physical parameter, or both.

[0180] In certain embodiments, the compositions of the present inventionare administered to a subject as a preventative measure against suchcancer or an infectious disease. As used herein, “prevention” or“preventing” refers to a reduction of the risk of acquiring a givencancer or infectious disease. In one mode of the embodiment, thecompositions of the present invention are administered as a preventativemeasure to a subject having a genetic predisposition to a cancer. Inanother mode of the embodiment, the compositions of the presentinvention are administered as a preventive measure to a subject facingexposure to carcinogens including but not limited to chemicals and/orradiation, or to a subject facing exposure to an agent of an infectiousdisease.

[0181] For example, in certain embodiments, administration of thecompositions of the invention leads to an inhibition or reduction of thegrowth of cancerous cells or infectious agents by at least 99%, at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 60%, at least 50%, at least 45%, at least 40%, at least45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least10% relative to the growth in absence of said composition.

[0182] The compositions prepared by methods of the invention comprisecomplexes of heat shock protein(s) with a population of antigenicpeptides, and/or complexes of alpha-2-macroglobulin with a population ofantigenic peptides. The compositions appear to induce an inflammatoryreaction at the tumor site and can ultimately cause a regression of thetumor burden in the cancer patients treated. The compositions preparedby the methods of the invention can enhance the immunocompetence of thesubject and elicit specific immunity against infectious agents orspecific immunity against preneoplastic and neoplastic cells. Thesecompositions have the capacity to prevent the onset and progression ofinfectious diseases, and to inhibit the growth and progression of tumorcells.

[0183] Combination therapy refers to the use of HSP complexes or α2Mcomplexes of the invention with another modality to prevent or treatcancer and infectious diseases. The administration of the complexes ofthe invention can augment the effect of anti-cancer agents oranti-infectives, and vice versa. Preferably, this additional form ofmodality is a non-HSP and non-α2M based modality, i.e., this modalitydoes not comprise either HSP or α2M as a component. This approach iscommonly termed combination therapy, adjunctive therapy or conjunctivetherapy (the terms are used interchangeably herein). With combinationtherapy, additive potency or additive therapeutic effect can beobserved. Synergistic outcomes where the therapeutic efficacy is greaterthan additive can also be expected. The use of combination therapy canalso provide better therapeutic profiles than the administration of thetreatment modality, or the HSP complexes or α2M complexes alone. Theadditive or synergistic effect may allow the dosage and/or dosingfrequency of either or both modalities be adjusted to reduce or avoidunwanted or adverse effects.

[0184] In various specific embodiments, the combination therapycomprises the administration of HSP complexes or α2M complexes to asubject treated with a treatment modality wherein the treatment modalityadministered alone is not clinically adequate to treat the subject suchthat the subject needs additional effective therapy, e.g., a subject isunresponsive to a treatment modality without administering HSP complexesor α2M complexes. Included in such embodiments are methods comprisingadministering HSP complexes or α2M complexes to a subject receiving atreatment modality wherein said subject has responded to therapy yetsuffers from side effects, relapse, develops resistance, etc. Such asubject might be non-responsive or refractory to treatment with thetreatment modality alone, i.e., at least some significant portion ofcancer cells or pathogens are not killed or their cell division is notarrested. The embodiments provide that the methods of the inventioncomprising administration of HSP complexes to a subject refractory to atreatment modality alone can improve the therapeutic effectiveness ofthe treatment modality when administered as contemplated by the methodsof the invention. The methods of the invention comprising administrationof an α2M complexes to a subject refractory to a treatment modalityalone can also improve the therapeutic effectiveness of the treatmentmodality when administered as contemplated by the methods of theinvention. The determination of the effectiveness of a treatmentmodality can be assayed in vivo or in vitro using methods known in theart. Art-accepted meanings of refractory are well known in the contextof cancer. In one embodiment, a cancer or infectious disease isrefractory or non-responsive where respectively, the number of cancercells or pathogens has not been significantly reduced, or has increased.Among these subjects being treated are those receiving chemotherapy orradiation therapy.

[0185] According to the invention, complexes of the invention can beused in combination with many different types of treatment modalities.Some of such modalities are particularly useful for a specific type ofcancer or infectious disease and are discussed in Section 4.5.1 and4.5.2. Many other modalities have an effect on the functioning of theimmune system and are applicable generally to both neoplastic andinfectious diseases .

[0186] In one embodiment, complexes of the invention are used incombination with one or more biological response modifiers to treatcancer or infectious disease. One group of biological response modifiersis the cytokines. In one such embodiment, a cytokine is administered toa subject receiving HSP/α2M complexes. In another such embodiment,HSP/α2M complexes are administered to a subject receiving achemotherapeutic agent in combination with a cytokine. In variousembodiments, one or more cytokine(s) can be used and are selected fromthe group consisting of IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IFNα, IFNβ, IFNγ, TNFα, TNFβ,G-CSF, GM-CSF, TGF-β, IL-15, IL-18, GM-CSF, INF-γ, INF-α, SLC,endothelial monocyte activating protein-2 (EMAP2), MIP-3α, MIP-3β, or anMHC gene, such as HLA-B7. Addtionally, other exemplary cytokines includeother members of the TNF family, including but not limited toTNF-α-related apoptosis-inducing ligand (TRAIL), TNF-α-relatedactivation-induced cytokine (TRANCE), TNF-α-related weak inducer ofapoptosis (TWEAK), CD40 ligand (CD40L), lymphotoxin alpha (LT-α),lymphotoxin beta (LT-β), OX40 ligand (OX40L), Fas ligand (FasL), CD27ligand (CD27L), CD30 ligand (CD30L), 41BB ligand (41BBL), APRIL, LIGHT,TL1, TNFSF16, TNFSF17, and AITR-L, or a functional portion thereof. See,e.g., Kwon et al., 1999, Curr. Opin. Immunol. 11:340-345 for a generalreview of the TNF family. Preferably, the HSP complexes or α2M complexesis administered prior to the treatment modalities. In a specificembodiment, complexes of the invention are administered to a subjectreceiving cyclophosphamide in combination with IL-12 for treatment ofcancer.

[0187] In another embodiments, complexes of the invention are used incombination with one or more biological response modifiers which areagonists or antagonists of various ligands, receptors and signaltransduction molecules of the immune system. For examples, thebiological response modifiers include but are not limited to agoinsts ofToll-like receptors (TLR-2, TLR-7, TLR-8 and TLR-9; LPS; agonists of41BB ligand, OX40 ligand, ICOS, and CD40; and antagonists of Fas ligand,PD1, and CTLA-4. These agonists and antagonists can be antibodies,antibody fragments, peptides, peptidomimetic compounds, andpolysaccharides.

[0188] In yet another embodiment, complexes of the invention are used incombination with one or more biological response modifiers which areimmunostimulatory nucleic acids. Such nucleic acids, many of which areoligonucleotides comprising an unmethylated CpG motif, are mitogenic tovertebrate lymphocytes, and are known to enhance the immune response.See Woolridge, et al., 1997, Blood 89:2994-2998. Such oligonucleotidesare described in International Patent Publication Nos. WO 01/22972, WO01/51083, WO 98/40100 and WO 99/61056, each of which is incorporatedherein in its entirety, as well as U.S. Pat. Nos. 6,207,646, 6,194,388,6,218,371, 6,239,116, 6,429,199, and 6,406,705, each of which isincorporated herein in its entirety. Other kinds of immunostimulatoryoligonucleotides such as phosphorothioate oligodeoxynucleotidescontaining YpG- and CpR-motifs have been described by Kandimalla et al.in “Effect of Chemical Modifications of Cytosine and Guanine in aCpG-Motif of Oligonucleotides: Structure-Immunostimulatory ActivityRelationships.” Bioorganic & Medicinal Chemistry 9:807-813 (2001),incorporated herein by reference in its entirety. Also encompassed areimmunostimulatory oligonucleotides that lack CpG dinucleotides whichwhen administered by mucosal routes (including low dose administration)or at high doses through parenteral routes, augment antibody responses,often as much as did the CpG nucleic acids, however the response wasTh2-biased (IgG1>>IgG2a). See U.S. Patent Publication No. 20010044416A1, which is incorporated herein by reference in its entirety. Methodsof determining the activity of immunostimulatory oligonucleotides can beperformed as described in the aforementioned patents and publications.Moreover, immunostimulatory oligonucleotides can be modified within thephosphate backbone, sugar, nucleobase and intenucleotide linkages inorder to modulate the activity. Such modifications are known to those ofskill in the art.

[0189] In yet another embodiment, complexes of the invention are used incombination with one or more adjuvants. The adjuvant(s) can beadministered separately or present in a composition in admixture withcomplexes of the invention. A systemic adjuvant is an adjuvant that canbe delivered parenterally. Systemic adjuvants include adjuvants thatcreates a depot effect, adjuvants that stimulate the immune system andadjuvants that do both. An adjuvant that creates a depot effect as usedherein is an adjuvant that causes the antigen to be slowly released inthe body, thus prolonging the exposure of immune cells to the antigen.This class of adjuvants includes but is not limited to alum (e.g.,aluminum hydroxide, aluminum phosphate); or emulsion-based formulationsincluding mineral oil, non-mineral oil, water-in-oil or oil-in-water-inoil emulsion, oil-in-water emulsions such as Seppic ISA series ofMontanide adjuvants (e.g., Montanide ISA 720, AirLiquide, Paris,France); MF-59 (a squalene-in-water emulsion stabilized with Span 85 andTween 80; Chiron Corporation, Emeryville, Calif.; and PROVAX (anoil-in-water emulsion containing a stabilizing detergent and amicelle-forming agent; IDEC, Pharmaceuticals Corporation, San Diego,Calif.).

[0190] Other adjuvants stimulate the immune system, for instance, causean immune cell to produce and secrete cytokines or IgG. This class ofadjuvants includes but is not limited to immunostimulatory nucleicacids, such as CpG oligonucleotides; saponins purified from the bark ofthe Q. saponaria tree, such as QS21;poly[di(carboxylatophen-oxy)phosphazene (PCPP polymer; Virus ResearchInstitute, USA); derivatives of lipopolysaccharides (LPS) such asmonophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton,Mont.), muramyl dipeptide (MDP; Ribi) andthreonyl-muramyl dipeptide(t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A; OMPharma SA, Meyrin, Switzerland); and Leishmania elongation factor (apurified Leishmania protein; Corixa Corporation, Seattle, Wash.).

[0191] Other systemic adjuvants are adjuvants that create a depot effectand stimulate the immune system. These compounds are those compoundswhich have both of the above-identified functions of systemic adjuvants.This class of adjuvants includes but is not limited to ISCOMs(Immunostimulating complexes which contain mixed saponins, lipids andform virus-sized particles with pores that can hold antigen; CSL,Melbourne, Australia); SB-AS2 (SmithKline Beecham adjuvant system #2which is an oil-in-water emulsion containing MPL and QS21: SmithKlineBeecham Biologicals [SBB], Rixensart, Belgium); SB-AS4 (SmithKlineBeecham adjuvant system #4 which contains alum and MPL; SBB, Belgium);non-ionic block copolymers that form micelles such as CRL 1005 (thesecontain a linear chain of hydrophobic polyoxpropylene flanked by chainsof polyoxyethylene; Vaxcel, Inc., Norcross, Ga.); and Syntex AdjuvantFormulation (SAF, an oil-in-water emulsion containing Tween 80 and anonionic block copolymer; Syntex Chemicals, Inc., Boulder, Colo.).

[0192] The mucosal adjuvants useful according to the invention areadjuvants that are capable of inducing a mucosal immune response in asubject when administered to a mucosal surface in conjunction withcomplexes of the invention. Mucosal adjuvants include but are notlimited to CpG nucleic acids (e.g. PCT published patent application WO99/61056), Bacterial toxins: e.g., Cholera toxin (CT), CT derivativesincluding but not limited to CT B subunit (CTB) (Wu et al., 1998,Tochikubo et al., 1998); CTD53 (Val to Asp) (Fontana et al., 1995);CTK97 (Val to Lys) (Fontana et al., 1995); CTK104 (Tyr to Lys) (Fontanaet al., 1995); CTD53/K63 (Val to Asp, Ser to Lys) (Fontana et al.,1995); CTH54 (Arg to His) (Fontana et al., 1995); CTN107 (His to Asn)(Fontana et al., 1995); CTE114 (Ser to Glu) (Fontana et al., 1995);CTE112K (Glu to Lys) (Yamamoto et al., 1997a); CTS61F (Ser to Phe)(Yamamoto et al., 1997a, 1997b); CTS106 (Pro to Lys) (Douce et al.,1997, Fontana et al., 1995); and CTK63 (Ser to Lys) (Douce et al., 1997,Fontana et al., 1995), Zonula occludens toxin, zot, Escherichia coliheat-labile enterotoxin, Labile Toxin (LT), LT derivatives including butnot limited to LT B subunit (LTB) (Verweij et al., 1998); LT7K (Arg toLys) (Komase et al., 1998, Douce et al., 1995); LT61F (Ser to Phe)(Komase et al., 1998); LT112K (Glu to Lys) (Komase et al., 1998); LT118E(Gly to Glu) (Komase et al., 1998); LT146E (Arg to Glu) (Komase et al.,1998); LT192G (Arg to Gly) (Komase et al., 1998); LTK63 (Ser to Lys)(Marchetti et al., 1998, Douce et al., 1997, 1998, Di Tommaso et al.,1996); and LTR72 (Ala to Arg) (Giuliani et al., 1998), Pertussis toxin,PT. (Lycke et al., 1992, Spangler BD, 1992, Freytag and Clemments, 1999,Roberts et al., 1995, Wilson et al., 1995) including PT-9K/129G (Robertset al., 1995, Cropley et al., 1995); Toxin derivatives (see below)(Holmgren et al., 1993, Verweij et al., 1998, Rappuoli et al., 1995,Freytag and Clements, 1999); Lipid A derivatives (e.g., monophosphoryllipid A, MPL) (Sasaki et al., 1998, Vancott et al., 1998; MuramylDipeptide (MDP) derivatives (Fukushima et al., 1996, Ogawa et al., 1989,Michalek et al., 1983, Morisaki et al., 1983); bacterial outer membraneproteins (e.g., outer surface protein A (OspA) lipoprotein of Borreliaburgdorferi, outer membrane protine of Neisseria meningitidis)(Marinaroet al., 1999, Van de Verg et al., 1996); oil-in-water emulsions (e.g.,MF59) (Barchfield et al., 1999, Verschoor et al., 1999, O'Hagan, 1998);aluminum salts (Isaka et al., 1998, 1999); and Saponins (e.g., QS21)Aquila Biopharmaceuticals, Inc., Worster, Me.) (Sasaki et al., 1998,MacNeal et al., 1998), ISCOMs, MF-59 (a squalene-in-water emulsionstabilized with Span 85 and Tween 80; Chiron Corporation, Emeryville,Calif.); the Seppic ISA series of Montanide adjuvants (e.g., MontanideISA 720; AirLiquide, Paris, France); PROVAX (an oil-in-water emulsioncontaining a stabilizing detergent and a micell-forming agent; IDECPharmaceuticals Corporation, San Diego, Calif.); Syntext AdjuvantFormulation (SAF; Syntex Chemicals, Inc., Boulder, Colo.);poly[di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus ResearchInstitute, USA) and Leishmania elongation factor (Corixa Corporation,Seattle, Wash.).

[0193] 4.5.1. Target Cancers

[0194] In one embodiment, combination therapy encompasses, in additionto the administration of the complexes of the invention, the adjunctiveuses of one or more modalities that aid in the prevention or treatmentof cancer, which modalities include, but is not limited tochemotherapeutic agents, immunotherapeutics, anti-angiogenic agents,cytokines, hormones, antibodies, polynucleotides, radiation andphotodynamic therapeutic agents. In specific embodiments, combinationtherapy can be used to prevent the recurrence of cancer, inhibitmetastasis, or inhibit the growth and/or spread of cancer or metastasis.

[0195] Types of cancers that can be treated or prevented by the methodsof the present invention include, but are not limited to human sarcomasand carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acutemyelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic,monocytic and erythroleukemia); chronic leukemia (chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia); andpolycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin'sdisease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavychain disease.

[0196] In another embodiment, the patient having a cancer isimmunosuppressed by reason of having undergone anti-cancer therapy(e.g., chemotherapy radiation) prior to administration of the HSP and/orα2M-peptide complexes or administration of the HSP-and/or α2M-sensitizedAPC.

[0197] There are many reasons why immunotherapy as provided by thepresent invention is desired for use in cancer patients. First, surgerywith anesthesia may lead to immunosuppression. With appropriateimmunotherapy in the preoperative period, this immunosuppression may beprevented or reversed. This could lead to fewer infectious complicationsand to accelerated wound healing. Second, tumor bulk is minimalfollowing surgery and immunotherapy is most likely to be effective inthis situation. A third reason is the possibility that tumor cells areshed into the circulation at surgery and effective immunotherapy appliedat this time can eliminate these cells.

[0198] The preventive and therapeutic methods of the invention aredirected at enhancing the immunocompetence of the cancer patient eitherbefore surgery, at or after surgery, and to induce tumor-specificimmunity to cancer cells, with the objective being inhibition of cancer,and with the ultimate clinical objective being total cancer regressionand eradication. The methods of the invention can also be used inindividuals at enhanced risk of a particular type of cancer, e.g., dueto familial history or environmental risk factors.

[0199] In various embodiments, one or more anti-cancer agent, inaddition to the complexes of the invention, is administered to treat acancer patient. An anti-cancer agent refers to any molecule or compoundthat assists in the treatment of tumors or cancer. Examples ofanti-cancer agents that may be used in the methods of the presentinvention include, but are not limited to: acivicin; aclarubicin;acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine;ambomycin; ametantrone acetate; aminoglutethimide; amsacrine;anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa;azotomycin; batimastat; benzodepa; bicalutamide; bisantrenehydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate;brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone;caracemide; carbetimer; carboplatin; carmustine; carubicinhydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin;cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine;dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine;dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel;doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifenecitrate; dromostanolone propionate; duazomycin; edatrexate; eflomithinehydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine;epirubicin hydrochloride; erbulozole; esorubicin hydrochloride;estramustine; estramustine phosphate sodium; etanidazole; etoposide;etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine;fenretinide; floxuridine; fludarabine phosphate; fluorouracil;flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabinehydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide;ilmofosine; interleukin II (including recombinant interleukin II, orrIL2), interferon alfa-2a; interferon alfa-2b; interferon alfa-n1;interferon alfa-n3; interferon beta-I a; interferon gamma-I b;iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole;leuprolide acetate; liarozole hydrochloride; lometrexol sodium;lomustine; losoxantrone hydrochloride; masoprocol; maytansine;mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate;melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium;metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin;mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride;mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran;paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride;semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermaniumhydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin;sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantronehydrochloride; temoporfin; teniposide; teroxirone; testolactone;thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifenecitrate; trestolone acetate; triciribine phosphate; trimetrexate;trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracilmustard; uredepa; vapreotide; verteporfin; vinblastine sulfate;vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate;vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;zinostatin; zorubicin hydrochloride.

[0200] Other anti-cancer drugs that can be used include, but are notlimited to: 20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil;abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin;aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox;amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide;anastrozole; andrographolide; angiogenesis inhibitors; antagonist D;antagonist G; antarelix; anti-dorsalizing morphogenetic protein-1;antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston;antisense oligonucleotides; aphidicolin glycinate; apoptosis genemodulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA;arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1;axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatinIII derivatives; balanol; batimastat; BCR/ABL antagonists;benzochlorins; benzoylstaurosporine; beta lactam derivatives;beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor;bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistrateneA; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine;calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2;capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRestM3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinaseinhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorlns;chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine;clomifene analogues; clotrimazole; collismycin A; collismycin B;combretastatin A4; combretastatin analogue; conagenin; crambescidin 816;crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A;cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate;cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B;deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;diaziquone; didemnin B; didox; diethylnorspermine;dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenylspiromustine; docetaxel; docosanol; dolasetron; doxifluridine;droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine;edelfosine; edrecolomab; eflomithine; elemene; emitefur; epirubicin;epristeride; estramustine analogue; estrogen agonists; estrogenantagonists; etanidazole; etoposide phosphate; exemestane; fadrozole;fazarabine; fenretinide; filgrastim; finasteride; flavopiridol;flezelastine; fluasterone; fludarabine; fluorodaunorunicinhydrochloride; forfenimex; formestane; fostriecin; fotemustine;gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam;heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid;idarubicin; idoxifene; idramantone; ilmofosine; ilomastat;imidazoacridones; imiquimod; immunostimulant peptides; insulin-likegrowth factor-1 receptor inhibitor; interferon agonists; interferons;interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact;irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustardanti-cancer agent; mycaperoxide B; mycobacterial cell wall extract;myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin;nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim;nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase;nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant;nitrullyn; O6-benzylguanine; octreotide; okicenone; oligonucleotides;onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer;ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxelanalogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin;pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine;pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin;pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras famesyl protein transferase inhibitors; rasinhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B 1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen binding protein; sizofiran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatinstimalamer.

[0201] An anti-cancer agent can be a chemotherapeutic agents whichinclude but are not limited to, the following groups of compounds :cytotoxic antibiotics, antimetabolities, anti-mitotic agents, alkylatingagents, platinum compounds, arsenic compounds, DNA topoisomeraseinhibitors, taxanes, nucleoside analogues, plant alkaloids, and toxins;and synthetic derivatives thereof. Table 1 lists exemplary compounds ofthe groups: TABLE 1 Alkylating agents Nitrogen mustards:Cyclophosphamide Ifosfamide Trofosfamide Chlorambucil Nitrosoureas:Carmustine (BCNU) Lomustine (CCNU) Alkylsulphonates: Busulfan TreosulfanTriazenes: Dacarbazine Platinum containing Cisplatin compounds:Carboplatin Aroplatin Oxaliplatin Plant Alkaloids Vinca alkaloids:Vincristine Vinblastine Vindesine Vinorelbine Taxoids: PaclitaxelDocetaxol DNA Topoisomerase Inhibitors Epipodophyllins: EtoposideTeniposide Topotecan 9-aminocamptothecin Camptothecin Crisnatolmitomycins: Mitomycin C Anti-folates: DHFR inhibitors: MethotrexateTrimetrexate IMP dehydrogenase Mycophenolic acid Inhibitors: TiazofurinRibavirin EICAR Ribonuclotide reductase Hydroxyurea Inhibitors:Deferoxamine Pyrimidine analogs: Uracil analogs: 5-FluorouracilFloxuridine Doxifluridine Ratitrexed Cytosine analogs: Cytarabine (araC) Cytosine arabinoside Fludarabine Purine analogs: MercaptopurineThioguanine DNA Antimetabolites: 3-HP 2′-deoxy-5-fluorouridine 5-HPalpha-TGDR aphidicolin glycinate ara-C 5-aza-2′-deoxycytidine beta-TGDRcyclocytidine guanazole inosine glycodialdehyde macebecin IIpyrazoloimidazole Antimitotic agents: allocolchicine Halichondrin Bcolchicine colchicine derivative dolstatin 10 maytansine rhizoxinthiocolchicine trityl cysteine Others: Isoprenylation inhibitors:Dopaminergic neurotoxins: 1-methyl-4-phenylpyridinium ion Cell cycleinhibitors: Staurosporine Actinomycins: Actinomycin D DactinomycinBleomycins: Bleomycin A2 Bleomycin B2 Peplomycin Anthracyclines:Daunorubicin Doxorubicin (adriamycin) Idarubicin Epirubicin PirarubicinZorubicin Mitoxantrone MDR inhibitors: Verapamil Ca²⁺ ATPase inhibitors:Thapsigargin

[0202] Compositions comprising one or more chemotherapeutic agents(e.g., FLAG, CHOP) are also contemplated by the present invention. FLAGcomprises fludarabine, cytosine arabinoside (Ara-C) and G-CSF. CHOPcomprises cyclophosphamide, vincristine, doxorubicin, and prednisone.Each of the foregoing lists is illustrative, and is not intended to belimiting.

[0203] In one embodiment, breast cancer can be treated with apharmaceutical composition comprising complexes of the invention incombination with 5-fluorouracil, cisplatin, docetaxel, doxorubicin,Herceptin®, gemcitabine, IL-2, paclitaxel, and/or VP-16 (etoposide).

[0204] In another embodiment, prostate cancer can be treated with apharmaceutical composition comprising complexes of the invention incombination with paclitaxel, docetaxel, mitoxantrone, and/or an androgenreceptor antagonist (e.g., flutamide).

[0205] In another embodiment, leukemia can be treated with apharmaceutical composition comprising complexes of the invention incombination with fludarabine, cytosine arabinoside, gemtuzumab(MYLOTARG), daunorubicin, methotrexate, vincristine, 6-mercaptopurine,idarubicin, mitoxantrone, etoposide, asparaginase, prednisone and/orcyclophosphamide. As another example, myeloma can be treated with apharmaceutical composition comprising complexes of the invention incombination with dexamethasone. Preferably, the leukemia is chronicmyeloid leukemia (CML), the HSP complexes comprises hsp70-peptidecomplexes, and the therapeutic modality is imatinib mesylate orGleevec™.

[0206] In another embodiment, melanoma can be treated with apharmaceutical composition comprising complexes of the invention incombination with dacarbazine.

[0207] In another embodiment, colorectal cancer can be treated with apharmaceutical composition comprising complexes of the invention incombination with irinotecan.

[0208] In another embodiment, lung cancer can be treated with apharmaceutical composition comprising complexes of the invention incombination with paclitaxel, docetaxel, etoposide and/or cisplatin.

[0209] In another embodiment, non-Hodgkin's lymphoma can be treated witha pharmaceutical composition comprising complexes of the invention incombination with cyclophosphamide, CHOP, etoposide, bleomycin,mitoxantrone and/or cisplatin.

[0210] In another embodiment, gastric cancer can be treated with apharmaceutical composition comprising complexes of the invention incombination with cisplatin.

[0211] In another embodiment, pancreatic cancer can be treated with apharmaceutical composition comprising complexes of the invention incombination with gemcitabine.

[0212] According to the invention, the complexes of the invention can beadministered prior to, subsequently, or concurrently with anti-canceragent(s), for the prevention or treatment of cancer. Depending on thetype of cancer, the subject's history and condition, and the anti-canceragent(s) of choice, the use of the complexes of the invention can becoordinated with the dosage and timing of chemotherapy.

[0213] The use of the complexes of the invention can be added to aregimen of chemotherapy. In one embodiment, the chemotherapeutic agentis gemcitabine at a dose ranging from 100 to 1000 mg/m²/cycle. In oneembodiment, the chemotherapeutic agent is dacarbazine at a dose rangingfrom 200 to 4000 mg/m²/cycle. In a preferred embodiment, the dose ofdacarbazine ranges from 700 to 1000 mg/m²/cycle. In another embodiment,the chemotherapeutic agent is fludarabine at a dose ranging from 25 to50 mg/m²/cycle. In another embodiment, the chemotherapeutic agent iscytosine arabinoside (Ara-C) at a dose ranging from 200 to 2000mg/m²/cycle. In another embodiment, the chemotherapeutic agent isdocetaxel at a dose ranging from 1.5 to 7.5 mg/kg/cycle. In anotherembodiment, the chemotherapeutic agent is paclitaxel at a dose rangingfrom 5 to 15 mg/kg/cycle. In yet another embodiment, thechemotherapeutic agent is cisplatin at a dose ranging from 5 to 20mg/kg/cycle. In yet another embodiment, the chemotherapeutic agent is5-fluorouracil at a dose ranging from 5 to 20 mg/kg/cycle. In yetanother embodiment, the chemotherapeutic agent is doxorubicin at a doseranging from 2 to 8 mg/kg/cycle. In yet another embodiment, thechemotherapeutic agent is epipodophyllotoxin at a dose ranging from 40to 160 mg/kg/cycle. In yet another embodiment, the chemotherapeuticagent is cyclophosphamide at a dose ranging from 50 to 200 mg/kg/cycle.In yet another embodiment, the chemotherapeutic agent is irinotecan at adose ranging from 50 to 75, 75 to 100, 100 to 125, or 125 to 150mg/m²/cycle. In yet another embodiment, the chemotherapeutic agent isvinblastine at a dose ranging from 3.7 to 5.4, 5.5 to 7.4, 7.5 to 11, or11 to 18.5 mg/m²/cycle. In yet another embodiment, the chemotherapeuticagent is vincristine at a dose ranging from 0.7 to 1.4, or 1.5 to 2 mg/m²/cycle. In yet another embodiment, the chemotherapeutic agent ismethotrexate at a dose ranging from 3.3 to 5, 5 to 10, 10 to 100, or 100to 1000 mg/m²/cycle.

[0214] In a preferred embodiment, the invention further encompasses theuse of low doses of chemotherapeutic agents when administered as part ofthe combination therapy regimen. For example, initial treatment with thecomplexes of the invention increases the sensitivity of a tumor tosubsequent challenge with a dose of chemotherapeutic agent, which doseis near or below the lower range of dosages when the chemotherapeuticagent is administered without complexes of the invention.

[0215] In one embodiment, complexes of the invention and a low dose(e.g., 6 to 60 mg/m²/day or less) of docetaxel are administered to acancer patient. In another embodiment, complexes of the invention and alow dose (e.g., 10 to 135 mg/m²/day or less) of paclitaxel areadministered to a cancer patient. In yet another embodiment, complexesof the invention and a low dose (e.g., 2.5 to 25 mg/m²/day or less) offludarabine are administered to a cancer patient. In yet anotherembodiment, complexes of the invention and a low dose (e.g., 0.5 to 1.5g/m²/day or less) of cytosine arabinoside (Ara-C) are administered to acancer patient. In another embodiment, the chemotherapeutic agent isgemcitabine at a dose ranging from 10 to 100 mg/m²/cycle. In anotherembodiment, the chemotherapeutic agent is cisplatin, e.g., PLATINOL orPLATINOL-AQ (Bristol Myers), at a dose ranging from 5 to 10, 10 to 20,20 to 40, or 40 to 75 mg/m²/cycle. In yet another embodiment, a dose ofcisplatin ranging from 7.5 to 75 mg/m²/cycle is administered to apatient with ovarian cancer. In yet another embodiment, a dose ofcisplatin ranging from 5 to 50 mg/m²/cycle is administered to a patientwith bladder cancer. In yet another embodiment, the chemotherapeuticagent is carboplatin, e.g., PARAPLATIN (Bristol Myers), at a doseranging from 2 to 4, 4 to 8, 8 to 16, 16 to 35, or 35 to 75 mg/m²/cycle.In yet another embodiment, a dose of carboplatin ranging from 7.5 to 75mg/m²/cycle is administered to a patient with ovarian cancer. In anotherembodiment, a dose of carboplatin ranging from 5 to 50 mg/m²/cycle isadministered to a patient with bladder cancer. In yet anotherembodiment, a dose of carboplatin ranging from 2 to 20 mg/m²/cycle isadministered to a patient with testicular cancer. In yet anotherembodiment, the chemotherapeutic agent is docetaxel, e.g., TAXOTERE(Rhone Poulenc Rorer) at a dose ranging from 6 to 10, 10 to 30, or 30 to60 mg/m²/cycle. In yet another embodiment, the chemotherapeutic agent ispaclitaxel, e.g., TAXOL (Bristol Myers Squibb), at a dose ranging from10 to 20, 20 to 40, 40 to 70, or 70 to 135 mg/kg/cycle. In yet anotherembodiment, the chemotherapeutic agent is 5-fluorouracil at a doseranging from 0.5 to 5 mg/kg/cycle. In yet another embodiment, thechemotherapeutic agent is doxorubicin, e.g., ADRIAMYCIN (Pharmacia &Upjohn), DOXIL (Alza), RUBEX (Bristol Myers Squibb), at a dose rangingfrom 2 to 4, 4 to 8, 8 to 15, 15 to 30, or 30 to 60 mg/kg/cycle.

[0216] In another embodiment, complexes of the invention is administeredin combination with one or more immunotherapeutic agents, such asantibodies and vaccines. In a preferred embodiment, the antibodies havein vivo therapeutic and/or prophylactic uses against cancer. In someembodiments, the antibodies can be used for treatment and/or preventionof infectious disease. Examples of therapeutic and prophylacticantibodies include, but are not limited to, MDX-010 (Medarex, NJ) whichis a humanized anti-CTLA-4 antibody currently in clinic for thetreatment of prostate cancer; SYNAGIS(® (MedImmune, MD) which is ahumanized anti-respiratory syncytial virus (RSV) monoclonal antibody forthe treatment of patients with RSV infection; HERCEPTIN® (Trastuzumab)(Genentech, CA) which is a humanized anti-HER2 monoclonal antibody forthe treatment of patients with metastatic breast cancer. Other examplesare a humanized anti-CD18 F(ab′)₂ (Genentech); CDP860 which is ahumanized anti-CD18 F(ab′)₂ (Celltech, UK); PRO542 which is an anti-HIVgp120 antibody fused with CD4 (Progenics/Genzyme Transgenics); Ostavirwhich is a human anti Hepatitis B virus antibody (Protein DesignLab/Novartis); PROTOVIR™ which is a humanized anti-CMV IgG1 antibody(Protein Design Lab/Novartis); MAK-195 (SEGARD) which is a murineanti-TNF-α F(ab′)₂ (Knoll Pharma/BASF); IC14 which is an anti-CD14antibody (ICOS Pharm); a humanized anti-VEGF IgG1 antibody (Genentech);OVAREX™ which is a murine anti-CA 125 antibody (Altarex); PANOREX™ whichis a murine anti-17-IA cell surface antigen IgG2a antibody (GlaxoWellcome/Centocor); BEC2 which is a murine anti-idiotype (GD3 epitope)IgG antibody (ImClone System); IMC-C225 which is a chimeric anti-EGFRIgG antibody (ImClone System); VITAXIN™ which is a humanized anti-αVβ3integrin antibody (Applied Molecular Evolution/MedImmune); Campath1H/LDP-03 which is a humanized anti CD52 IgG1 antibody (Leukosite);Smart M195 which is a humanized anti-CD33 IgG antibody (Protein DesignLab/Kanebo); RITUXAN™ which is a chimeric anti-CD20 IgG1 antibody (IDECPharm/Genentech, Roche/Zettyaku); LYMPHOCIDE™ which is a humanizedanti-CD22 IgG antibody (Immunomedics); Smart ID10 which is a humanizedanti-HLA antibody (Protein Design Lab); ONCOLYM™ (Lym-1) is aradiolabelled murine anti-HLA DIAGNOSTIC REAGENT antibody (Techniclone);ABX-IL8 is a human anti-IL8 antibody (Abgenix); anti-CD11 a is ahumanized IgG1 antibody (Genentech/Xoma); ICM3 is a humanized anti-ICAM3antibody (ICOS Pharm); IDEC-114 is a primatized anti-CD80 antibody (IDECPharm/Mitsubishi); ZEVALIN™ is a radiolabelled murine anti-CD20 antibody(IDEC/Schering AG); IDEC-131 is a humanized anti-CD40L antibody(IDEC/Eisai); IDEC-151 is a primatized anti-CD4 antibody (IDEC);IDEC-152 is a primatized anti-CD23 antibody (IDEC/Seikagaku); SMARTanti-CD3 is a humanized anti-CD3 IgG (Protein Design Lab); 5G1.1 is ahumanized anti-complement factor 5 (C5) antibody (Alexion Pharm); D2E7is a humanized anti-TNF-α antibody (CAT/BASF); CDP870 is a humanizedanti-TNF-α Fab fragment (Celltech); IDEC-151 is a primatized anti-CD4IgG1 antibody (IDEC Pharm/SmithKline Beecham); MDX-CD4 is a humananti-CD4 IgG antibody (Medarex/Eisai/Genmab); CDP571 is a humanizedanti-TNF-α IgG4 antibody (Celltech); LDP-02 is a humanized anti-α4β7antibody (LeukoSite/Genentech); OrthoClone OKT4A is a humanized anti-CD4IgG antibody (Ortho Biotech); ANTOVA™ is a humanized anti-CD40L IgGantibody (Biogen); ANTEGREN™ is a humanized anti-VLA-4 IgG antibody(Elan); MDX-33 is a human anti-CD64 (FcγR) antibody (Medarex/Centeon);SCH55700 is a humanized anti-IL-5 IgG4 antibody (Celltech/Schering);SB-240563 and SB-240683 are humanized anti-IL-5 and IL-4 antibodies,respectively, (SmithKline Beecham); rhuMab-E25 is a humanized anti-IgEIgG1 antibody (Genentech/Norvartis/Tanox Biosystems); ABX-CBL is amurine anti CD-147 IgM antibody (Abgenix); BTI-322 is a rat anti-CD2 IgGantibody (Medimmune/Bio Transplant); Orthoclone/OKT3 is a murineanti-CD3 IgG2a antibody (ortho Biotech); SIMULECT™ is a chimericanti-CD25 IgG1 antibody (Novartis Pharm); LDP-01 is a humanizedanti-β₂-integrin IgG antibody (LeukoSite); Anti-LFA-1 is a murine antiCD18 F(ab′)₂ (Pasteur-Merieux/Immunotech); CAT-152 is a humananti-TGF-β₂ antibody (Cambridge Ab Tech); and Corsevin M is a chimericanti-Factor VII antibody (Centocor). The above-listed immunoreactivereagents, as well as any other immunoreactive reagents, may beadministered according to any regimen known to those of skill in theart, including the regimens recommended by the suppliers of theimmunoreactive reagents.

[0217] In another embodiment, complexes of the invention is administeredin combination with one or more anti-angiogenic agents, which includes,but is not limited to, angiostatin, thalidomide, kringle 5, endostatin,Serpin (Serine Protease Inhibitor) anti-thrombin, 29 kDa N-terminal anda 40 kDa C-terminal proteolytic fragments of fibronectin, 16 kDaproteolytic fragment of prolactin, 7.8 kDa proteolytic fragment ofplatelet factor-4, a 13-amino acid peptide corresponding to a fragmentof platelet factor-4 (Maione et al., 1990, Cancer Res. 51:2077-2083), a14-amino acid peptide corresponding to a fragment of collagen I (Tolmaet al., 1993, J. Cell Biol. 122:497-511), a 19 amino acid peptidecorresponding to a fragment of Thrombospondin I (Tolsma et al., 1993, J.Cell Biol. 122:497-511), a 20-amino acid peptide corresponding to afragment of SPARC (Sage et al., 1995, J. Cell. Biochem. 57:1329-1334),or any fragments, family members, or variants thereof, includingpharmaceutically acceptable salts thereof.

[0218] Other peptides that inhibit angiogenesis and correspond tofragments of laminin, fibronectin, procollagen, and EGF have also beendescribed (see, e.g., Cao, 1998, Prog Mol Subcell Biol. 20:161-176).Monoclonal antibodies and cyclic pentapeptides, which block certainintegrins that bind RGD proteins (i.e., possess the peptide motifArg-Gly-Asp), have been demonstrated to have anti-vascularizationactivities (Brooks et al., 1994, Science 264:569-571; Hammes et al.,1996, Nature Medicine 2:529-533). Moreover, inhibition of the urokinaseplasminogen activator receptor by receptor antagonists inhibitsangiogenesis, tumor growth and metastasis (Min et al., 1996, Cancer Res.56: 2428-33; Crowley et al., 1993, Proc Natl Acad Sci. 90:5021-25). Useof such anti-angiogenic agents in combination with the complexes is alsocontemplated by the present invention.

[0219] In yet another embodiment, complexes of the invention is used inassociation with a hormonal treatment. Hormonal therapeutic treatmentscomprise hormonal agonists, hormonal antagonists (e.g., flutamide,bicalutamide, tamoxifen, raloxifene, leuprolide acetate (LUPRON), LH-RHantagonists), inhibitors of hormone biosynthesis and processing, andsteroids (e.g., dexamethasone, retinoids, deltoids, betamethasone,cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids,mineralocorticoids, estrogen, testosterone, progestins), vitamin Aderivatives (e.g., all-trans retinoic acid (ATRA)); vitamin D3 analogs;antigestagens (e.g., mifepristone, onapristone), and antiandrogens(e.g., cyproterone acetate).

[0220] In yet another embodiment, complexes of the invention are used inassociation with a gene therapy program in the treatment of cancer. Inone embodiment, gene therapy with recombinant cells secretinginterleukin-2 is administered in combination with complexes of theinvention to prevent or treat cancer, particularly breast cancer (See,e.g., Deshmukh et al., 2001, J Neurosurg. 94:287-92). In otherembodiments, gene therapy is conducted with the use of polynucleotidecompounds, such as but not limited to antisense polynucleotides,ribozymes, RNA interference molecules, triple helix polynucleotides andthe like, where the nucleotide sequence of such compounds are related tothe nucleotide sequences of DNA and/or RNA of genes that are linked tothe initiation, progression, and/or pathology of a tumor or cancer. Forexample, many are oncogenes, growth factor genes, growth factor receptorgenes, cell cycle genes, DNA repair genes, and are well known in theart.

[0221] In another embodiment, complexes of the invention is administeredin conjunction with a regimen of radiation therapy. For radiationtreatment, the radiation can be gamma rays or X-rays. The methodsencompass treatment of cancer comprising radiation therapy, such asexternal-beam radiation therapy, interstitial implantation ofradioisotopes (I-125, palladium, iridium), radioisotopes such asstrontium-89, thoracic radiation therapy, intraperitoneal P-32 radiationtherapy, and/or total abdominal and pelvic radiation therapy. For ageneral overview of radiation therapy, see Hellman, Chapter 16:Principles of Cancer Management: Radiation Therapy, 6th edition, 2001,DeVita et al., eds., J.B. Lippencott Company, Philadelphia. In preferredembodiments, the radiation treatment is administered as external beamradiation or teletherapy wherein the radiation is directed from a remotesource. In various preferred embodiments, the radiation treatment isadministered as internal therapy or brachytherapy wherein a radiaoactivesource is placed inside the body close to cancer cells or a tumor mass.Also encompassed is the combined use of complexes of the invention withphotodynamic therapy comprising the administration of photosensitizers,such as hematoporphyrin and its derivatives, Vertoporfin (BPD-MA),phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin A; and2BA-2-DMHA.

[0222] In various embodiments, complexes of the invention isadministered, in combination with at least one chemotherapeutic agent,for a short treatment cycle to a cancer patient to treat cancer. Theduration of treatment with the chemotherapeutic agent may vary accordingto the particular cancer therapeutic agent used. The invention alsocontemplates discontinuous administration or daily doses divided intoseveral partial administrations. An appropriate treatment time for aparticular cancer therapeutic agent will be appreciated by the skilledartisan, and the invention contemplates the continued assessment ofoptimal treatment schedules for each cancer therapeutic agent. Thepresent invention contemplates at least one cycle, preferably more thanone cycle during which a single therapeutic or sequence of therapeuticsis administered. An appropriate period of time for one cycle will beappreciated by the skilled artisan, as will the total number of cycles,and the interval between cycles.

[0223] In another embodiment, complexes of the invention are used incombination with compounds that ameliorate the symptoms of the cancer(such as but not limited to pain) and the side effects produced by thecomplexes of the invention (such as but not limited to flu-likesymptoms, fever, etc). Accordingly, many compounds known to reduce pain,flu-like symptoms, and fever can be used in combination or in admixturewith complexes of the invention. Such compounds include analgesics (e.g,acetaminophen), decongestants (e.g., pseudoephedrine), antihistamines(e.g., chlorpheniramine maleate), and cough suppressants (e.g.,dextromethorphan).

[0224] 4.5.2. Target Infectious Diseases

[0225] Infectious diseases that can be treated or prevented by themethods of the present invention are caused by infectious agentsincluding, but not limited to, viruses, bacteria, fungi protozoa,helminths, and parasites. The invention is not limited to treating orpreventing infectious diseases caused by intracellular pathogens. Manymedically relevant microorganisms have been described extensively in theliterature, e.g., see C.G.A Thomas, Medical Microbiology, BailliereTindall, Great Britain 1983, the entire contents of which is herebyincorporated by reference.

[0226] Combination therapy encompasses in addition to the administrationof complexes of the invention, the uses of one or more modalities thataid in the prevention or treatment of infectious diseases, whichmodalities include, but is not limited to antibiotics, antivirals,antiprotozoal compounds, antifungal compounds, and antihelminthics.Other treatment modalities that can be used to treat or preventinfectious diseases include immunotherapeutics, polynucleotides,antibodies, cytokines, and hormones as described above.

[0227] Infectious virus of both human and non-human vertebrates, includeretroviruses, RNA viruses and DNA viruses. Examples of virus that havebeen found in humans include but are not limited to: Retroviridae (e.g.human immunodeficiency viruses, such as HIV-1 (also referred to asHTLV-III, LAV or HTLV-III/LAV, or HIV-III; and other isolates, such asHIV-LP; Picomaviridae (e.g. polio viruses, hepatitis A virus;enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses);Calciviridae (e.g. strains that cause gastroenteritis); Togaviridae(e.g. equine encephalitis viruses, rubella viruses); Flaviridae (e.g.dengue viruses, encephalitis viruses, yellow fever viruses);Coronaviridae (e.g. coronaviruses); Rhabdoviridae (e.g. vesicularstomatitis viruses, rabies viruses); Filoviridae (e.g. ebola viruses);Paramyxoviridae (e.g. parainfluenza viruses, mumps virus, measles virus,respiratory syncytial virus); Orthomyxoviridae (e.g. influenza viruses);Bungaviridae (e.g. Hantaan viruses, bunga viruses, phleboviruses andNairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae(e.g. reoviruses, orbiviurses and rotaviruses); Birnaviridae;Hepadnaviridae (Hepatitis B virus); Parvovirida (parvoviruses);Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (mostadenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2,varicella zoster virus, cytomegalovirus (CMV), herpes virus; Poxviridae(variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g.African swine fever virus); and unclassified viruses (e.g. theetiological agents of Spongiform encephalopathies, the agent of deltahepatitis (thought to be a defective satellite of hepatitis B virus),the agents of non-A, non-B hepatitis (class 1=intemally transmitted;class 2=parenterally transmitted (i.e. Hepatitis C); Norwalk and relatedviruses, and astroviruses).

[0228] Retroviruses that are contemplated include both simpleretroviruses and complex retroviruses. The simple retroviruses includethe subgroups of B-type retroviruses, C-type retroviruses and D-typeretroviruses. An example of a B-type retrovirus is mouse mammary tumorvirus (MMTV). The C-type retroviruses include subgroups C-type group A(including Rous sarcoma virus (RSV), avian leukemia virus (ALV), andavian myeloblastosis virus (AMV)) and C-type group B (including murineleukemia virus (MLV), feline leukemia virus (FeLV), murine sarcoma virus(MSV), gibbon ape leukemia virus (GALV), spleen necrosis virus (SNV),reticuloendotheliosis virus (RV) and simian sarcoma virus (SSV)). TheD-type retroviruses include Mason-Pfizer monkey virus (MPMV) and simianretrovirus type 1 (SRV-1). The complex retroviruses include thesubgroups of lentiviruses, T-cell leukemia viruses and the foamyviruses. Lentiviruses include HIV-1, but also include HIV-2, SIV, Visnavirus, feline immunodeficiency virus (FIV), and equine infectious anemiavirus (EIAV). The T-cell leukemia viruses include HTLV-1, HTLV-II,simian T-cell leukemia virus (STLV), and bovine leukemia virus (BLV).The foamy viruses include human foamy virus (HFV), simian foamy virus(SFV) and bovine foamy virus (BFV).

[0229] Examples of RNA viruses that are antigens in vertebrate animalsinclude, but are not limited to, the following: members of the familyReoviridae, including the genus Orthoreovirus (multiple serotypes ofboth mammalian and avian retroviruses), the genus Orbivirus (Bluetonguevirus, Eugenangee virus, Kemerovo virus, African horse sickness virus,and Colorado Tick Fever virus), the genus Rotavirus (human rotavirus,Nebraska calf diarrhea virus, murine rotavirus, simian rotavirus, bovineor ovine rotavirus, avian rotavirus); the family Picornaviridae,including the genus Enterovirus (poliovirus, Coxsackie virus A and B,enteric cytopathic human orphan (ECHO) viruses, hepatitis A virus,Simian enteroviruses, Murine encephalomyelitis (ME) viruses, Poliovirusmuris, Bovine enteroviruses, Porcine enteroviruses, the genusCardiovirus (Encephalomyocarditis virus (EMC), Mengovirus), the genusRhinovirus (Human rhinoviruses including at least 113 subtypes; otherrhinoviruses), the genus Apthovirus (Foot and Mouth disease (FMDV); thefamily Calciviridae, including Vesicular exanthema of swine virus, SanMiguel sea lion virus, Feline picornavirus and Norwalk virus; the familyTogaviridae, including the genus Alphavirus (Eastern equine encephalitisvirus, Semliki forest virus, Sindbis virus, Chikungunya virus,O'Nyong-Nyong virus, Ross river virus, Venezuelan equine encephalitisvirus, Western equine encephalitis virus), the genus Flavirius (Mosquitoborne yellow fever virus, Dengue virus, Japanese encephalitis virus, St.Louis encephalitis virus, Murray Valley encephalitis virus, West Nilevirus, Kunjin virus, Central European tick borne virus, Far Eastern tickborne virus, Kyasanur forest virus, Louping III virus, Powassan virus,Omsk hemorrhagic fever virus), the genus Rubivirus (Rubella virus), thegenus Pestivirus (Mucosal disease virus, Hog cholera virus, Borderdisease virus); the family Bunyaviridae, including the genus Bunyvirus(Bunyamwera and related viruses, California encephalitis group viruses),the genus Phlebovirus (Sandfly fever Sicilian virus, Rift Valley fevervirus), the genus Nairovirus (Crimean-Congo hemorrhagic fever virus,Nairobi sheep disease virus), and the genus Uukuvirus (Uukuniemi andrelated viruses); the family Orthomyxoviridae, including the genusInfluenza virus (Influenza virus type A, many human subtypes); Swineinfluenza virus, and Avian and Equine Influenza viruses; influenza typeB (many human subtypes), and influenza type C (possible separate genus);the family paramyxoviridae, including the genus Paramyxovirus(Parainfluenza virus type 1, Sendai virus, Hemadsorption virus,Parainfluenza viruses types 2 to 5, Newcastle Disease Virus, Mumpsvirus), the genus Morbillivirus (Measles virus, subacute sclerosingpanencephalitis virus, distemper virus, Rinderpest virus), the genusPneumovirus (respiratory syncytial virus (RSV), Bovine respiratorysyncytial virus and Pneumonia virus of mice); forest virus, Sindbisvirus, Chikungunya virus, O'Nyong-Nyong virus, Ross river virus,Venezuelan equine encephalitis virus, Western equine encephalitisvirus), the genus Flavirius (Mosquito borne yellow fever virus, Denguevirus, Japanese encephalitis virus, St. Louis encephalitis virus, MurrayValley encephalitis virus, West Nile virus, Kunjin virus, CentralEuropean tick borne virus, Far Eastern tick borne virus, Kyasanur forestvirus, Louping III virus, Powassan virus, Omsk hemorrhagic fever virus),the genus Rubivirus (Rubella virus), the genus Pestivirus (Mucosaldisease virus, Hog cholera virus, Border disease virus); the familyBunyaviridae, including the genus Bunyvirus (Bunyamwera and relatedviruses, California encephalitis group viruses), the genus Phlebovirus(Sandfly fever Sicilian virus, Rift Valley fever virus), the genusNairovirus (Crimean-Congo hemorrhagic fever virus, Nairobi sheep diseasevirus), and the genus Uukuvirus (Uukuniemi and related viruses); thefamily Orthomyxoviridae, including the genus Influenza virus (Influenzavirus type A, many human subtypes); Swine influenza virus, and Avian andEquine Influenza viruses; influenza type B (many human subtypes), andinfluenza type C (possible separate genus); the family paramyxoviridae,including the genus Paramyxovirus (Parainfluenza virus type 1, Sendaivirus, Hemadsorption virus, Parainfluenza viruses types 2 to 5,Newcastle Disease Virus, Mumps virus), the genus Morbillivirus (Measlesvirus, subacute sclerosing panencephalitis virus, distemper virus,Rinderpest virus), the genus Pneumovirus (respiratory syncytial virus(RSV), Bovine respiratory syncytial virus and Pneumonia virus of mice);the family Rhabdoviridae, including the genus Vesiculovirus (VSV),Chandipura virus, Flanders-Hart Park virus), the genus Lyssavirus(Rabies virus), fish Rhabdoviruses, and two probable Rhabdoviruses(Marburg virus and Ebola virus); the family Arenaviridae, includingLymphocytic choriomeningitis virus (LCM), Tacaribe virus complex, andLassa virus; the family Coronoaviridae, including Infectious BronchitisVirus (IBV), Mouse Hepatitis virus, Human enteric corona virus, andFeline infectious peritonitis (Feline coronavirus).

[0230] Illustrative DNA viruses that are antigens in vertebrate animalsinclude, but are not limited to: the family Poxviridae, including thegenus Orthopoxvirus (Variola major, Variola minor, Monkey pox Vaccinia,Cowpox, Buffalopox, Rabbitpox, Ectromelia), the genus Leporipoxvirus(Myxoma, Fibroma), the genus Avipoxvirus (Fowlpox, other avianpoxvirus), the genus Capripoxvirus (sheeppox, goatpox), the genusSuipoxvirus (Swinepox), the genus Parapoxvirus (contagious postulardermatitis virus, pseudocowpox, bovine papular stomatitis virus); thefamily Iridoviridae (African swine fever virus, Frog viruses 2 and 3,Lymphocystis virus of fish); the family Herpesviridae, including thealpha-Herpesviruses (Herpes Simplex Types 1 and 2, Varicella-Zoster,Equine abortion virus, Equine herpes virus 2 and 3, pseudorabies virus,infectious bovine keratoconjunctivitis virus, infectious bovinerhinotracheitis virus, feline rhinotracheitis virus, infectiouslaryngotracheitis virus) the Beta-herpesviruses (Human cytomegalovirusand cytomegaloviruses of swine, monkeys and rodents); thegamma-herpesviruses (Epstein-Barr virus (EBV), Marek's disease virus,Herpes saimiri, Herpesvirus ateles, Herpesvirus sylvilagus, guinea pigherpes virus, Lucke tumor virus); the family Adenoviridae, including thegenus Mastadenovirus (Human subgroups A,B,C,D,E and ungrouped; simianadenoviruses (at least 23 serotypes), infectious canine hepatitis, andadenoviruses of cattle, pigs, sheep, frogs and many other species, thegenus Aviadenovirus (Avian adenoviruses); and non-cultivatableadenoviruses; the family Papoviridae, including the genus Papillomavirus(Human papilloma viruses, bovine papilloma viruses, Shope rabbitpapilloma virus, and various pathogenic papilloma viruses of otherspecies), the genus Polyomavirus (polyomavirus, Simian vacuolating agent(SV-40), Rabbit vacuolating agent (RKV), K virus, BK virus, JC virus,and other primate polyoma viruses such as Lymphotrophic papillomavirus); the family Parvoviridae including the genus Adeno-associatedviruses, the genus Parvovirus (Feline panleukopenia virus, bovineparvovirus, canine parvovirus, Aleutian mink disease virus, etc).Finally, DNA viruses may include viruses which do not fit into the abovefamilies such as Kuru and Creutzfeldt-Jacob disease viruses and chronicinfectious neuropathic agents.

[0231] Many examples of antiviral compounds that can be used incombination with the complexes of the invention are known in the art andinclude but are not limited to: rifampicin, nucleoside reversetranscriptase inhibitors (e.g., AZT, ddI, ddC, 3TC, d4T), non-nucleosidereverse transcriptase inhibitors (e.g., Efavirenz, Nevirapine), proteaseinhibitors (e.g., aprenavir, indinavir, ritonavir, and saquinavir),idoxuridine, cidofovir, acyclovir, ganciclovir, zanamivir, amantadine,and Palivizumab. Other examples of anti-viral agents include but are notlimited to Acemannan; Acyclovir; Acyclovir Sodium; Adefovir; Alovudine;Alvircept Sudotox; Amantadine Hydrochloride; Aranotin; Arildone;Atevirdine Mesylate; Avridine; Cidofovir; Cipamfylline; CytarabineHydrochloride; Delavirdine Mesylate; Desciclovir; Didanosine; Disoxaril;Edoxudine; Enviradene; Enviroxime; Famciclovir; Famotine Hydrochloride;Fiacitabine; Fialuridine; Fosarilate; Foscamet Sodium; Fosfonet Sodium;Ganciclovir; Ganciclovir Sodium; Idoxuridine; Kethoxal; Lamivudine;Lobucavir; Memotine Hydrochloride; Methisazone; Nevirapine; Penciclovir;Pirodavir; Ribavirin; Rimantadine Hydrochloride; Saquinavir Mesylate;Somantadine Hydrochloride; Sorivudine; Statolon; Stavudine; TiloroneHydrochloride; Trifluridine; Valacyclovir Hydrochloride; Vidarabine;Vidarabine Phosphate; Vidarabine Sodium Phosphate; Viroxime;Zalcitabine; Zidovudine; Zinviroxime.

[0232] Bacterial infections or diseases that can be treated or preventedby the methods of the present invention are caused by bacteriaincluding, but not limited to, bacteria that have an intracellular stagein its life cycle, such as mycobacteria (e.g., Mycobacteriatuberculosis, M. bovis, M avium, M leprae, or M. africanum), rickettsia,mycoplasma, chlamydia, and legionella. Other examples of bacterialinfections contemplated include but are not limited to infections causedby Gram positive bacillus (e.g., Listeria, Bacillus such as Bacillusanthracis, Erysipelothrix species), Gram negative bacillus (e.g.,Bartonella, Brucella, Campylobacter, Enterobacter, Escherichia,Francisella, Hemophilus, Klebsiella, Morganella, Proteus, Providencia,Pseudomonas, Salmonella, Serratia, Shigella, Vibrio, and Yersiniaspecies), spirochete bacteria (e.g., Borrelia species including Borreliaburgdorferi that causes Lyme disease), anaerobic bacteria (e.g.,Actinomyces and Clostridium species), Gram positive and negative coccalbacteria, Enterococcus species, Streptococcus species, Pneumococcusspecies, Staphylococcus species, Neisseria species. Specific examples ofinfectious bacteria include but are not limited to: Helicobacterpyloris,Borelia burgdorferi, Legionella pneumophilia, Mycobacteria tuberculosis,M. avium, M. intracellulare, M. kansaii, M. gordonae, Staphylococcusaureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeriamonocytogenes, Streptococcus pyogenes (Group A Streptococcus),Streptococcus agalactiae (Group B Streptococcus), Streptococcusviridans, Streptococcus faecalis, Streptococcus bovis, Streptococcuspneumoniae, Haemophilus influenzae, Bacillus antracis, corynebacteriumdiphtheriae, Erysipelothrix rhusiopathiae, Clostridium perfringers,Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae,Pasturella multocida, Fusobacterium nucleatum, Streptobacillusmoniliformis, Treponema pallidium, Treponema pertenue, Leptospira,Rickettsia, and Actinomyces israelli.

[0233] Antibacterial agents or antibiotics that can be used incombination with the complexes of the invention include but are notlimited to: aminoglycoside antibiotics (e.g., apramycin, arbekacin,bambermycins, butirosin, dibekacin, neomycin, neomycin, undecylenate,netilmicin, paromomycin, ribostamycin, sisomicin, and spectinomycin),amphenicol antibiotics (e.g., azidamfenicol, chloramphenicol,florfenicol, and thiamphenicol), ansamycin antibiotics (e.g., rifamideand rifampin), carbacephems (e.g., loracarbef), carbapenems (e.g.,biapenem and imipenem), cephalosporins (e.g., cefaclor, cefadroxil,cefamandole, cefatrizine, cefazedone, cefozopran, cefpimizole,cefpiramide, and cefpirome), cephamycins (e.g., cefbuperazone,cefmetazole, and cefminox), monobactams (e.g., aztreonam, carumonam, andtigemonam), oxacephems (e.g., flomoxef, and moxalactam), penicillins(e.g., amdinocillin, amdinocillin pivoxil, amoxicillin, bacampicillin,benzylpenicillinic acid, benzylpenicillin sodium, epicillin,fenbenicillin, floxacillin, penamccillin, penethamate hydriodide,penicillin o-benethamine, penicillin 0, penicillin V, penicillin Vbenzathine, penicillin V hydrabamine, penimepicycline, andphencihicillin potassium), lincosamides (e.g., clindamycin, andlincomycin), macrolides (e.g., azithromycin, carbomycin, clarithomycin,dirithromycin, erythromycin, and erythromycin acistrate), amphomycin,bacitracin, capreomycin, colistin, enduracidin, enviomycin,tetracyclines (e.g., apicycline, chlortetracycline, clomocycline, anddemeclocycline), 2,4-diaminopyrimidines (e.g., brodimoprim), nitrofurans(e.g., furaltadone, and furazolium chloride), quinolones and analogsthereof (e.g., cinoxacin, ciprofloxacin, clinafloxacin, flumequine, andgrepagloxacin), sulfonamides (e.g., acetyl sulfamethoxypyrazine,benzylsulfamide, noprylsulfamide, phthalylsulfacetamide,sulfachrysoidine, and sulfacytine), sulfones (e.g., diathymosulfone,glucosulfone sodium, and solasulfone), cycloserine, mupirocin andtuberin.

[0234] Additional examples of antibacterial agents include but are notlimited to Acedapsone; Acetosulfone Sodium; Alamecin; Alexidine;Amdinocillin; Amdinocillin Pivoxil; Amicycline; Amifloxacin; AmifloxacinMesylate; Amikacin; Amikacin Sulfate; Aminosalicylic acid;Aminosalicylate sodium; Amoxicillin; Amphomycin; Ampicillin; AmpicillinSodium; Apalcillin Sodium; Apramycin; Aspartocin; Astromicin Sulfate;Avilamycin; Avoparcin; Azithromycin; Azlocillin; Azlocillin Sodium;Bacampicillin Hydrochloride; Bacitracin; Bacitracin MethyleneDisalicylate; Bacitracin Zinc; Bambermycins; Benzoylpas Calcium;Berythromycin; Betamicin Sulfate; Biapenem; Biniramycin; BiphenamineHydrochloride; Bispyrithione Magsulfex; Butikacin; Butirosin Sulfate;Capreomycin Sulfate; Carbadox; Carbenicillin Disodium; CarbenicillinIndanyl Sodium; Carbenicillin Phenyl Sodium; Carbenicillin Potassium;Carumonam Sodium; Cefaclor; Cefadroxil; Cefamandole; Cefamandole Nafate;Cefamandole Sodium; Cefaparole; Cefatrizine; Cefazaflur Sodium;Cefazolin; Cefazolin Sodium; Cefbuperazone; Cefdinir; Cefepime; CefepimeHydrochloride; Cefetecol; Cefixime; Cefmnenoxime Hydrochloride;Cefinetazole; Cefmetazole Sodium; Cefonicid Monosodium; CefonicidSodium; Cefoperazone Sodium; Ceforanide; Cefotaxime Sodium; Cefotetan;Cefotetan Disodium; Cefotiam Hydrochloride; Cefoxitin; Cefoxitin Sodium;Cefpimizole; Cefpimizole Sodium; Cefpiramide; Cefpiramide Sodium;Cefpirome Sulfate; Cefpodoxime Proxetil; Cefprozil; Cefroxadine;Cefsulodin Sodium; Ceftazidime; Ceftibuten; Ceftizoxime Sodium;Ceftriaxone Sodium; Cefuroxime; Cefuroxime Axetil; Cefuroxime Pivoxetil;Cefuroxime Sodium; Cephacetrile Sodium; Cephalexin; CephalexinHydrochloride; Cephaloglycin; Cephaloridine; Cephalothin Sodium;Cephapirin Sodium; Cephradine; Cetocycline Hydrochloride; Cetophenicol;Chloramphenicol; Chloramphenicol Palmitate; Chloramphenicol PantothenateComplex; Chloramphenicol Sodium Succinate; Chlorhexidine Phosphanilate;Chloroxylenol; Chlortetracycline Bisulfate; ChlortetracyclineHydrochloride; Cinoxacin; Ciprofloxacin; Ciprofloxacin Hydrochloride;Cirolemycin; Clarithromycin; Clinafloxacin Hydrochloride; Clindamycin;Clindamycin Hydrochloride; Clindamycin Palmitate Hydrochloride;Clindamycin Phosphate; Clofazimine; Cloxacillin Benzathine; CloxacillinSodium; Cloxyquin; Colistimethate Sodium; Colistin Sulfate; Coumermycin;Coumermycin Sodium; Cyclacillin; Cycloserine; Dalfopristin; Dapsone;Daptomycin; Demeclocycline; Demeclocycline Hydrochloride; Demecycline;Denofungin; Diaveridine; Dicloxacillin; Dicloxacillin Sodium;Dihydrostreptomycin Sulfate; Dipyrithione; Dirithromycin; Doxycycline;Doxycycline Calcium; Doxycycline Fosfatex; Doxycycline Hyclate; DroxacinSodium; Enoxacin; Epicillin; Epitetracycline Hydrochloride;Erythromycin; Erythromycin Acistrate; Erythromycin Estolate;Erythromycin Ethylsuccinate; Erythromycin Gluceptate; ErythromycinLactobionate; Erythromycin Propionate; Erythromycin Stearate; EthambutolHydrochloride; Ethionamide; Fleroxacin; Floxacillin; Fludalanine;Flumequine; Fosfomycin; Fosfomycin Tromethamine; Fumoxicillin;Furazolium Chloride; Furazolium Tartrate; Fusidate Sodium; Fusidic Acid;Gentamicin Sulfate; Gloximonam; Gramicidin; Haloprogin; Hetacillin;Hetacillin Potassium; Hexedine; Ibafloxacin; Imipenem; Isoconazole;Isepamicin; Isoniazid; Josamycin; Kanamycin Sulfate; Kitasamycin;Levofuraltadone; Levopropylcillin Potassium; Lexithromycin; Lincomycin;Lincomycin Hydrochloride; Lomefloxacin; Lomefloxacin Hydrochloride;Lomefloxacin Mesylate; Loracarbef; Mafenide; Meclocycline; MeclocyclineSulfosalicylate; Megalomicin Potassium Phosphate; Mequidox; Meropenem;Methacycline; Methacycline Hydrochloride; Methenamine; MethenamineHippurate; Methenamine Mandelate; Methicillin Sodium; Metioprim;Metronidazole Hydrochloride; Metronidazole Phosphate; Mezlocillin;Mezlocillin Sodium; Minocycline; Minocycline Hydrochloride; MirincamycinHydrochloride; Monensin; Monensin Sodium; Nafcillin Sodium; NalidixateSodium; Nalidixic Acid; Natamycin; Nebramycin; Neomycin Palmitate;Neomycin Sulfate; Neomycin Undecylenate; Netilmicin Sulfate;Neutramycin; Nifuradene; Nifuraldezone; Nifuratel; Nifuratrone;Nifurdazil; Nifurimide; Nifurpirinol; Nifurquinazol; Nifurthiazole;Nitrocycline; Nitrofurantoin; Nitromide; Norfloxacin; Novobiocin Sodium;Ofloxacin; Ormetoprim; Oxacillin Sodium; Oximonam; Oximonam Sodium;Oxolinic Acid; Oxytetracycline; Oxytetracycline Calcium; OxytetracyclineHydrochloride; Paldimycin; Parachlorophenol; Paulomycin; Pefloxacin;Pefloxacin Mesylate; Penamecillin; Penicillin G Benzathine; Penicillin GPotassium; Penicillin G Procaine; Penicillin G Sodium; Penicillin V;Penicillin V Benzathine; Penicillin V Hydrabamine; Penicillin VPotassium; Pentizidone Sodium; Phenyl Aminosalicylate; PiperacillinSodium; Pirbenicillin Sodium; Piridicillin Sodium; PirlimycinHydrochloride; Pivampicillin Hydrochloride; Pivampicillin Pamoate;Pivampicillin Probenate; Polymyxin B Sulfate; Porfiromycin; Propikacin;Pyrazinamide; Pyrithione Zinc; Quindecamine Acetate; Quinupristin;Racephenicol; Ramoplanin; Ranimycin; Relomycin; Repromicin; Rifabutin;Rifametane; Rifamexil; Rifamide; Rifampin; Rifapentine; Rifaximin;Rolitetracycline; Rolitetracycline Nitrate; Rosaramicin; RosaramicinButyrate; Rosaramicin Propionate; Rosaramicin Sodium Phosphate;Rosaramicin Stearate; Rosoxacin; Roxarsone; Roxithromycin; Sancycline;Sanfetrinem Sodium; Sarmoxicillin; Sarpicillin; Scopafingin; Sisomicin;Sisomicin Sulfate; Sparfloxacin; Spectinomycin Hydrochloride;Spiramycin; Stallimycin Hydrochloride; Steffimycin; StreptomycinSulfate; Streptonicozid; Sulfabenz; Sulfabenzamide; Sulfacetamide;Sulfacetamide Sodium; Sulfacytine; Sulfadiazine; Sulfadiazine Sodium;Sulfadoxine; Sulfalene; Sulfamerazine; Sulfameter; Sulfamethazine;Sulfamethizole; Sulfamethoxazole; Sulfamonomethoxine; Sulfamoxole;Sulfanilate Zinc; Sulfanitran; Sulfasalazine; Sulfasomizole;Sulfathiazole; Sulfazamet; Sulfisoxazole; Sulfisoxazole Acetyl;Sulfisoxazole Diolamine; Sulfomyxin; Sulopenem; Sultamicillin; SuncillinSodium; Talampicillin Hydrochloride; Teicoplanin; TemafloxacinHydrochloride; Temocillin; Tetracycline; Tetracycline Hydrochloride;Tetracycline Phosphate Complex; Tetroxoprim; Thiamphenicol;Thiphencillin Potassium; Ticarcillin Cresyl Sodium; TicarcillinDisodium; Ticarcillin Monosodium; Ticlatone; Tiodonium Chloride;Tobramycin; Tobramycin Sulfate; Tosufloxacin; Trimethoprim; TrimethoprimSulfate; Trisulfapyrimidines; Troleandomycin; Trospectomycin Sulfate;Tyrothricin; Vancomycin; Vancomycin Hydrochloride; Virginiamycin;Zorbamycin.

[0235] Fungal diseases that can be treated or prevented by the methodsof the present invention include but not limited to aspergilliosis,crytococcosis, sporotrichosis, coccidioidomycosis,paracoccidioidomycosis, histoplasmosis, blastomycosis, zygomycosis, andcandidiasis.

[0236] Antifungal compounds that can be used in combination with thecomplexes of the invention include but are not limited to: polyenes(e.g., amphotericin b, candicidin, mepartricin, natamycin, andnystatin), allylamines (e.g., butenafine, and naftifine), imidazoles(e.g., bifonazole, butoconazole, chlordantoin, flutrimazole,isoconazole, ketoconazole, and lanoconazole), thiocarbamates (e.g.,tolciclate, tolindate, and tolnaftate), triazoles (e.g., fluconazole,itraconazole, saperconazole, and terconazole), bromosalicylchloranilide,buclosamide, calcium propionate, chlorphenesin, ciclopirox, azaserine,griseofulvin, oligomycins, neomycin undecylenate, pyrrolnitrin,siccanin, tubercidin, and viridin. Additional examples of antifungalcompounds include but are not limited to Acrisorcin; Ambruticin;Amphotericin B; Azaconazole; Azaserine; Basifungin; Bifonazole;Biphenamine Hydrochloride; Bispyrithione Magsulfex; ButoconazoleNitrate; Calcium Undecylenate; Candicidin; Carbol-Fuchsin; Chlordantoin;Ciclopirox; Ciclopirox Olamine; Cilofungin; Cisconazole; Clotrimazole;Cuprimyxin; Denofungin; Dipyrithione; Doconazole; Econazole; EconazoleNitrate; Enilconazole; Ethonam Nitrate; Fenticonazole Nitrate; Filipin;Fluconazole; Flucytosine; Fungimycin; Griseofulvin; Hamycin;Isoconazole; Itraconazole; Kalafungin; Ketoconazole; Lomofingin;Lydimycin; Mepartricin; Miconazole; Miconazole Nitrate; Monensin;Monensin Sodium; Naftifine Hydrochloride; Neomycin Undecylenate;Nifuratel; Nifurmerone; Nitralamine Hydrochloride; Nystatin; OctanoicAcid; Orconazole Nitrate; Oxiconazole Nitrate; Oxifungin Hydrochloride;Parconazole Hydrochloride; Partricin; Potassium Iodide; Proclonol;Pyrithione Zinc; Pyrrolnitrin; Rutamycin; Sanguinarium Chloride;Saperconazole; Scopafungin; Selenium Sulfide; Sinefungin; SulconazoleNitrate; Terbinafine; Terconazole; Thiram; Ticlatone; Tioconazole;Tolciclate; Tolindate; Tolnaftate; Triacetin; Triafuigin; UndecylenicAcid; Viridoflilvin; Zinc Undecylenate; and Zinoconazole Hydrochloride.

[0237] Parasitic diseases that can be treated or prevented by themethods of the present invention including, but not limited to,amebiasis, malaria, leishmania, coccidia, giardiasis, cryptosporidiosis,toxoplasmosis, and trypanosomiasis. Also encompassed are infections byvarious worms, such as but not limited to ascariasis, ancylostomiasis,trichuriasis, strongyloidiasis, toxoccariasis, trichinosis,onchocerciasis. filaria, and dirofilariasis. Also encompassed areinfections by various flukes, such as but not limited toschistosomiasis, paragonimiasis, and clonorchiasis. Parasites that causethese diseases can be classified based on whether they are intracellularor extracellular. An “intracellular parasite” as used herein is aparasite whose entire life cycle is intracellular. Examples of humanintracellular parasites include Leishmania spp., Plasmodium spp.,Trypanosoma cruzi, Toxoplasma gondii, Babesia spp., and Trichinellaspiralis. An “extracellular parasite” as used herein is a parasite whoseentire life cycle is extracellular. Extracellular parasites capable ofinfecting humans include Entamoeba histolytica, Giardia lamblia,Enterocytozoon bieneusi, Naegleria and Acanthamoeba as well as mosthelminths. Yet another class of parasites is defined as being mainlyextracellular but with an obligate intracellular existence at a criticalstage in their life cycles. Such parasites are referred to herein as“obligate intracellular parasites”. These parasites may exist most oftheir lives or only a small portion of their lives in an extracellularenvironment, but they all have at least one obligate intracellular stagein their life cycles. This latter category of parasites includesTrypanosoma rhodesiense and Trypanosoma gambiense, Isospora spp.,Cryptosporidium spp, Eimeria spp., Neospora spp., Sarcocystis spp., andSchistosoma spp.

[0238] Many examples of antiprotozoal compounds that can be used incombination with the complexes of the invention to treat parasiticdiseases are known in the art and include but are not limited to:quinines, chloroquine, mefloquine, proguanil, pyrimethamine,metronidazole, diloxanide furoate, tinidazole, amphotericin, sodiumstibogluconate, trimoxazole, and pentamidine isetionate. Many examplesof antiparasite drugs that can be used in combination with the complexesof the invention to treat parasitic diseases are known in the art andinclude but are not limited to: mebendazole, levamisole, niclosamide,praziquantel, albendazole, ivermectin, diethylcarbamazine, andthiabendazole. Further examples of anti-parasitic compounds include butare not limited to Acedapsone; Amodiaquine Hydrochloride; Amquinate;Arteflene; Chloroquine; Chloroquine Hydrochloride; ChloroquinePhosphate; Cycloguanil Pamoate; Enpiroline Phosphate; HalofantrineHydrochloride; Hydroxychloroquine Sulfate; Mefloquine Hydrochloride;Menoctone; Mirincamycin Hydrochloride; Primaquine Phosphate;Pyrimethamine; Quinine Sulfate; and Tebuquine.

[0239] In a less preferred embodiment, the complexes of the inventioncan be used in combination with a non-HSP and non-α2M-based vaccinecomposition. Examples of such vaccines for humans are described in TheJordan Report 2000, Accelerated Development of Vaccines, NationalInstitute of Health, which is incorporated herein by reference in itsentirety. Many vaccines for the treatment of non-human vertebrates aredisclosed in Bennett, K. Compendium of Veterinary Products, 3rd ed.North American Compendiums, Inc., 1995, which is incorporated herein byreference in its entirety.

[0240] 4.5.3. Autologous Embodiment

[0241] The specific immunogenicity of HSPs and α2M derives not from HSPsor α2M per se, but from the antigenic proteins and/or peptides bound tothem. In a preferred embodiment of the invention, the complexes in thecompositions of the inventions for use as cancer vaccines are autologouscomplexes, thereby circumventing two of the most intractable hurdles tocancer immunotherapy. First is the possibility that human cancers, likecancers of experimental animals, are antigenically distinct. Tocircumvent this hurdle, in a preferred embodiment of the presentinvention, the HSPs and/or α2M are complexed to antigenic proteins andpeptides, and the complexes are used to treat the cancers in the samesubject from which the proteins or peptides are derived. Second, mostcurrent approaches to cancer immunotherapy focus on determining theCTL-recognized epitopes of cancer cell lines. This approach requires theavailability of cell lines and CTLs against cancers. These reagents areunavailable for an overwhelming proportion of human cancers. In anembodiment of the present invention directed to the use of autologousantigenic proteins and/or peptides, cancer immunotherapy does not dependon the availability of cell lines or CTLs nor does it require definitionof the antigenic epitopes of cancer cells. These advantages makecomplexes of HSPs and/or α2M bound to autologous antigenic proteinsand/or peptides attractive immunogens against cancer.

[0242] In other embodiments, the antigenic peptides in the therapeuticor prophylactic complexes can be prepared from cancerous tissue of thesame type of cancer from a subject allogeneic to the subject to whom thecomplexes are administered.

4.6. Adoptive Immunotherapy

[0243] Adoptive immunotherapy refers to a therapeutic approach fortreating cancer or infectious diseases in which immune cells areadministered to a host with the aim that the cells mediate eitherdirectly or indirectly specific immunity to tumor cells and/or antigeniccomponents or regression of the tumor or treatment of infectiousdiseases, as the case may be. (See e.g., U.S. Pat. No. 5,985,270, issuedNov. 16, 1999, which is incorporated by reference herein in itsentirety).

[0244] In one embodiment, antigen presenting cells (APC) for use inadoptive immunotherapy are sensitized with HSPs and/or α2M complexedwith antigenic proteins and peptides prepared in accordance with themethods described herein. The complexes can be produced by complexingheat shock protein or alpha-2-macroglobulin to antigenic proteins thatare derived from at least 50% of the different proteins or at least 100different proteins present in antigenic cells or viral particles thatexpress an antigenic determinant of an agent that causes the infectiousdisease. The complexes can also be produced by (a) subjecting a proteinpreparation derived from cells of said type of cancer to eitherdigestion with a protease or contact with ATP, guanidium hydrochloride,and/or acid, to generate a population of antigenic peptides, and (b)complexing the population of antigenic peptides to heat shock protein oralpha-2-macroglobulin.

[0245] In another embodiment, therapy by administration of in vitrocomplexed antigenic peptides and HSPs and/or α2M prepared by the methodsof the invention may be combined with adoptive immunotherapy using APCsensitized by HSP- and/or α2M-antigenic peptide complexes prepared byany method known in the art (see e.g., U.S. Pat. No. 5,985,270) in whichthe antigenic peptides display the desired antigenicity (e.g., of thetype of cancer or pathogen). The sensitized APC can be administeredalone, in combination with the in vitro complexed proteins/peptides andHSPs and/or α2M, or before or after administration of the complexedproteins/peptides and HSPs and/or α2M. In particular, the use ofsensitized APC to prevent and treat cancer can further compriseadministering to the subject an amount, effective for said treatment orprevention, of complexes comprising heat shock protein and/oralpha-2-macroglobulin, complexed to antigenic proteins/peptides, whereinsaid complexes were produced as described above. Similarly, the use ofsensitized APC in treating or preventing a type of infectious disease,can further comprise administering to the subject an amount, effectivefor said treatment or prevention, of complexes comprising heat shockprotein and/or alpha-2-macroglobulin, complexed to antigenicproteins/peptides.

[0246] Furthermore, the mode of administration of the in vitro complexedantigenic proteins/peptides and HSPs and/or α2M can be varied, includingbut not limited to, e.g., subcutaneously, intravenously orintramuscularly, although intradermally is preferred. In anotherspecific embodiment, adoptive immunotherapy by administration of theantigen presenting cells sensitized with complexes made according to thepresent invention can be combined with therapy by administration by HSP-and/or α2M-antigenic molecule (e.g., peptide) complexes prepared by anymethod known in the art (see e.g., U.S. Pat. Nos. 5,750,119, 5,837,251,5,961,979, 5,935,576, PCT publications WO 94/14976 or WO 99/50303) inwhich the antigenic molecules display the desired antigenicity (e.g., ofthe type of cancer or pathogen).

[0247] 4.6.1. Obtaining Antigen-Presenting Cells

[0248] The antigen-presenting cells, including but not limited tomacrophages, dendritic cells and B-cells, are preferably obtained byproduction in vitro from stem and progenitor cells from human peripheralblood or bone marrow as described by Inaba, K., et al., 1992, J. Exp.Med. 176:1693-1702. Dendritic cells can be obtained by any of variousmethods known in the art. By way of example but not limitation,dendritic cells can be obtained by the methods described in Sallusto etal., 1994, J Exp Med 179:1109-1118 and Caux et al., 1992, Nature 360,258-261 which are incorporated herein by reference in their entireties.In a preferred aspect, human dendritic cells obtained from human bloodcells are used.

[0249] APC can be obtained by any of various methods known in the art.In one aspect, human macrophages are used, obtained from human bloodcells. By way of example but not limitation, macrophages can be obtainedas follows:

[0250] Mononuclear cells are isolated from peripheral blood of a patient(preferably the patient to be treated), by Ficoll-Hypaque gradientcentrifugation and are seeded on tissue culture dishes which arepre-coated with the patient's own serum or with other AB+ human serum.The cells are incubated at 37° C. for 1 hour, then non-adherent cellsare removed by pipetting. To the adherent cells left in the dish, isadded cold (4° C.) 1 mM EDTA in phosphate-buffered saline and the dishesare left at room temperature for 15 minutes. The cells are harvested,washed with RPMI buffer and suspended in RPMI buffer. Increased numbersof macrophages may be obtained by incubating at 37° C. withmacrophage-colony stimulating factor (M-CSF).

[0251] 4.6.2. Sensitization of Macrophages and Antigen Presenting Cellswith HSP-Peptide or α2M-Peptide Complexes

[0252] APC are sensitized with HSP or α2M bound to antigenic peptidespreferably by incubating the cells in vitro with the complexes. The APCare sensitized with complexes of HSPs or α2M and antigenic molecules byincubating in vitro with the HSP-complex or α2M-complex at 37° C. for 15minutes to 24 hours. By way of example but not limitation, 4×10⁷dendritic cells can be incubated with 10 microgram gp96-peptidecomplexes per ml or 100 microgram HSP90-peptide complexes per ml at 37°C. for 15 minutes-24 hours in 1 ml plain RPMI medium. The cells arewashed three times and resuspended in a physiological medium preferablysterile, at a convenient concentration (e.g., 1×10⁷/ml) for injection ina patient. Preferably, the patient into which the sensitized dendriticcells are injected is the patient from which the dendritic cells wereoriginally isolated (autologous embodiment).

[0253] Optionally, the ability of sensitized APC to stimulate, forexample, the antigen-specific, class I-restricted cytotoxicT-lymphocytes (CTL) can be monitored by their ability to stimulate CTLsto release tumor necrosis factor, and by their ability to act as targetsof such CTLs.

[0254] 4.6.3. Reinfusion of Sensitized APC

[0255] The sensitized APCs are reinfused into the patient systemically,preferably intradermally, by conventional clinical procedures. Theseactivated cells are reinfused, preferentially by systemic administrationinto the autologous patient. Patients generally receive from about 10⁶to about 10¹² sensitized dendritic cells depending on the condition ofthe patient. In some regimens, patients may optionally receive inaddition a suitable dosage of a biological response modifier includingbut not limited to the cytokines IFN-α, IFN-γ, IL-2, IL-4, IL-6, TNF orother cytokine growth factor.

4.7. Pharmaceutical Preparations and Methods of Administration

[0256] The complexes of antigenic proteins/peptides bound to HSPs and/orα2M prepared by the methods of the invention can be administered to apatient at therapeutically effective doses to treat or ameliorate a cellproliferative disorder or infectious disease. A therapeuticallyeffective dose refers to that amount of the complexes sufficient toresult in amelioration of symptoms of such a disorder. The effectivedose of the complexes may be different when another treatment modalityis being used in combination. The appropriate and recommended dosages,formulation and routes of administration for treatment modalities suchas chemotherapeutic agents, radiation therapy andbiological/immunotherapeutic agents such as cytokines are known in theart and described in such literature as the Physician 's Desk Reference(56^(th) ed., 2002).

[0257] 4.7.1. Effective Dose

[0258] The compositions of the present invention, comprising animmunogenic, effective amount of complexes of a population of antigenicpeptides with HSP and/or α2M are administered to a subject in need oftreatment against cancer or an infectious disease, as a method ofinducing an immune response against that cancer or infectious disease.Toxicity and therapeutic efficacy of such complexes can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Complexes that exhibit large therapeutic indices are preferred. Whilecomplexes that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such complexes to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

[0259] In one embodiment, the data obtained from the cell culture assaysand animal studies can be used in formulating a range of dosage for usein humans. The dosage of complexes lies preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycomplexes used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

[0260] In another embodiment, an amount of hsp70- and/or gp96-antigenicmolecule complexes is administered that is in the range of about 0.1microgram to about 600 micrograms, and preferably about 1 micrograms toabout 60 micrograms for a human patient. The amount of hsp70- and/orgp96 complexes administered is 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 200, 250, 300, 400, 500 or 600 micrograms.Preferably, the amount is less than 100 micrograms. Most preferably, theamount of hsp70-and/or gp96 complexes administered is 5 micrograms, 25micrograms, or 50 micrograms. The dosage for hsp-90 peptide complexes ina human patient provided by the present invention is in the range ofabout 5 to 5,000 micrograms. Preferably, the the amount of hsp90complexes administered is 5, 10, 25, 50, 60, 70, 80, 90, 100, 200, 250,500, 1000, 2000, 2500, or 5000 microgram, the most preferred dosagebeing 100 microgram. These doses are preferably administeredintradermally or subcutaneously. These doses can be given once orrepeatedly, such as daily, every other day, weekly, biweekly, ormonthly. Preferably, the complexes are given once weekly for a period ofabout 4-6 weeks, and the mode or site of administration is preferablyvaried with each administration. Thus, by way of example and notlimitation, the first injection may be given subcutaneously on the leftarm, the second on the right arm, the third on the left belly, thefourth on the right belly, the fifth on the left thigh, the sixth on theright thigh, etc. The same site may be repeated after a gap of one ormore injections. Also, split injections may be given. Thus, for example,half the dose may be given in one site and the other half on an othersite on the same day. Alternatively, the mode of administration issequentially varied, e.g., weekly injections are given in sequenceintradermally, intramuscularly, subcutaneously, intravenously orintraperitoneally. Preferably, the once weekly dose is given for aperiod of 4 weeks. After 4-6 weeks, further injections are preferablygiven at two-week intervals over a period of time of one or more months,or until supply of complexes is exhausted. Later injections may be givenmonthly. The pace of later injections may be modified, depending uponthe patient's clinical progress and responsiveness to the immunotherapy.In a preferred example, intradermal administrations are given, with eachsite of administration varied sequentially.

[0261] Accordingly, the invention provides methods of preventing andtreating cancer or an infectious disease in a subject comprisingadministering a composition which stimulates the immunocompetence of thehost individual and elicits specific immunity against the preneoplasticand/or neoplastic cells or infected cells.

[0262] In a specific embodiment, during combination therapy, the HSPcomplexes is administered in a sub-optimal amount, e.g., an amount thatdoes not manifest detectable therapeutic benefits when administered inthe absence of the therapeutic modality, as determined by methods knownin the art. In such methods, the administration of such a sub-optimalamount of HSP complexes to a subject receiving a therapeutic modalityresults in an overall improvement in effectiveness of treatment. Inanother specific embodiment, the α2M complexes is administered in asub-optimal amount during combination therapy. In such methods, theadministration of such a sub-optimal amount of α2M complexes to asubject receiving a therapeutic modality results in an overallimprovement in effectiveness of treatment.

[0263] In a preferred embodiment, an HSP complexes is administered in anamount that does not result in tumor regression or cancer remission oran amount wherein the cancer cells have not been significantly reducedor have increased when said HSP complexes is administered in the absenceof the therapeutic modality. In a preferred embodiment, the sub-optimalamount of HSP complexes is administered to a subject receiving atreatment modality whereby the overall effectiveness of treatment isimproved. In another preferred embodiment, an α2M complexes isadministered in an amount that does not result in tumor regression orcancer remission or an amount wherein the cancer cells have not beensignificantly reduced or have increased when said α2M complexes isadministered in the absence of the therapeutic modality. In a preferredembodiment, the sub-optimal amount of α2M complexes is administered to asubject receiving a treatment modality whereby the overall effectivenessof treatment is improved. Among these subjects being treated with HSP orα2M complexes are those receiving chemotherapy or radiation therapy. Asub-optimal amount can be determined by appropriate animal studies. Sucha sub-optimal amount in humans can be determined by extrapolation fromexperiments in animals.

[0264] In certain specific embodiments, an HSP or α2M complexes isadministered to a subject already receiving a chemotherapeutic agent,such as Gleevec™ (e.g., 400-800 mg daily in capsule form, 400-600 mgdoses administered once daily, or 800 mg dose administered daily in twodoses of 400 mg each). Gleevec™ is used hereinbelow as a non-limitingexample of a chemotherapeutic agent that can be used in combination. Formany other chemotherapeutic agents, a similar dosing regime can be used.In such embodiments, the appropriate HSP/α2M complexes is initiallyadministered to a subject who has already been receiving Gleevec™ in theabsence of HSP/α2M complexes 2 days, 2 days to 1 week, 1 week to 1month, 1 month to 6 months, 6 months to 1 year prior to administrationof HSP/α2M complexes in addition to Gleevec™. In a specific embodiment,HSP/α2M complexes are administered to a subject wherein the subjectshowed resistance to treatment with Gleevec™ alone.

[0265] In other embodiments, HSP/α2M complexes are initiallyadministered to a subject concurrently with the initial administrationof Gleevec™.

[0266] In yet other specific embodiments, Gleevec™ (e.g., 400-800 mgdaily in capsule form) is administered to a subject already receivingtreatment comprising administration of HSP/α2M complexes. In suchembodiments, Gleevec™ is initially administered to a subject who hasalready been receiving HSP/α2M complexes in the absence of Gleevec™ 2days, 2 days to 1 week, 1 week to 1 month, 1 month to 6 months, 6 monthsto 1 year prior to administration of Gleevec™ in addition toadministration of HSP/α2M complexes.

[0267] In a specific embodiment, a chemotherapeutic agent such asGleevec™ is administered orally. In another specific embodiment, theHSP/α2M complexes are administered intradermally.

[0268] In each of the methods contemplated above, the subject, by way ofexample, receives 50 mg to 100 mg, 100 mg to 200 mg, 200 mg to 300 mg,300 mg to 400 mg, 400 mg to 500 mg, 500 mg to 600 mg, 600 mg to 700 mg,700 mg to 800 mg, 800 mg to 900 mg, or 900 mg to 1000 mg ofchemotherapeutic agents, such as Gleevec™, daily. In certainembodiments, the total daily dose is administered to a subject as twodaily doses of 25 mg to 50 mg, 50 mg to 100 mg, 100 mg to 200 mg, 200 mgto 300 mg, 300 mg to 400 mg, or 400 mg to 500 mg.

[0269] 4.7.2. Therapeutic Regimens

[0270] For any of the combination therapies described above fortreatment or prevention of cancer and infectious diseases, the complexesof the invention can be administered prior to, concurrently with, orsubsequent to the administration of the non-HSP and non-α2M basedmodality. The non-HSP and non-α2M based modality can be any one of themodalities described above for treatment or prevention of cancer orinfectious disease.

[0271] In one embodiment, the complexes of the invention is administeredto a subject at reasonably the same time as the other modality. Thismethod provides that the two administrations are performed within a timeframe of less than one minute to about five minutes, or up to aboutsixty minutes from each other, for example, at the same doctor's visit.

[0272] In another embodiment, the complexes of the invention and amodality are administered at exactly the same time. In yet anotherembodiment the complexes of the invention and the modality areadministered in a sequence and within a time interval such that thecomplexes of the invention and the modality can act together to providean increased benefit than if they were administered alone. In anotherembodiment, the complexes of the invention and a modality areadministered sufficiently close in time so as to provide the desiredtherapeutic or prophylactic outcome. Each can be administeredsimultaneously or separately, in any appropriate form and by anysuitable route. In one embodiment, the complexes of the invention andthe modality are administered by different routes of administration. Inan alternate embodiment, each is administered by the same route ofadministration. The complexes of the invention can be administered atthe same or different sites, e.g. arm and leg. When administeredsimultaneously, the complexes of the invention and the modality may ormay not be administered in admixture or at the same site ofadministration by the same route of administration.

[0273] In a preferred embodiment, the complexes of the invention areadministered according to the regimen described in Section 4.7.1. Invarious embodiments, the complexes of the invention and the modality areadministered less than 1 hour apart, at about 1 hour apart, 1 hour to 2hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hoursto 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7hours to 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hoursapart, 10 hours to 11 hours apart, 11 hours to 12 hours apart, no morethan 24 hours apart or no more than 48 hours apart. In otherembodiments, the complexes of the invention and vaccine composition areadministered 2 to 4 days apart, 4 to 6 days apart, 1 week a part, 1 to 2weeks apart, 2 to 4 weeks apart, one moth apart, 1 to 2 months apart, or2 or more months apart. In preferred embodiments, the complexes of theinvention and the modality are administered in a time frame where bothare still active. One skilled in the art would be able to determine sucha time frame by determining the half life of each administeredcomponent.

[0274] In one embodiment, the complexes of the invention and themodality are administered within the same patient visit. In a specificpreferred embodiment, the complexes of the invention is administeredprior to the administration of the modality. In an alternate specificembodiment, the complexes of the invention is administered subsequent tothe administration of the modality.

[0275] In certain embodiments, the complexes of the invention and themodality are cyclically administered to a subject. Cycling therapyinvolves the administration of the complexes of the invention for aperiod of time, followed by the administration of a modality for aperiod of time and repeating this sequential administration. Cyclingtherapy can reduce the development of resistance to one or more of thetherapies, avoid or reduce the side effects of one of the therapies,and/or improve the efficacy of the treatment. In such embodiments, theinvention contemplates the alternating administration of a complexes ofthe invention followed by the administration of a modality 4 to 6 dayslater, preferable 2 to 4 days, later, more preferably 1 to 2 days later,wherein such a cycle may be repeated as many times as desired. Incertain embodiments, the complexes of the invention and the modality arealternately administered in a cycle of less than 3 weeks, once every twoweeks, once every 10 days or once every week. In a specific embodiment,complexes of the invention is administered to a subject within a timeframe of one hour to twenty four hours after the administration of amodality. The time frame can be extended further to a few days or moreif a slow- or continuous-release type of modality delivery system isused.

[0276] 4.7.3. Formulations and Use

[0277] Pharmaceutical compositions for use in accordance with thepresent invention may be formulated in conventional manner using one ormore physiologically acceptable carriers or excipients.

[0278] Thus, the complexes and their physiologically acceptable saltsand solvates may be formulated for administration by inhalation orinsufflation (either through the mouth or the nose) oral, buccal,parenteral, rectal, or transdermal administration. Non-invasive methodsof administration are also contemplated.

[0279] For oral administration, the pharmaceutical compositions may takethe form of, for example, tablets or capsules prepared by conventionalmeans with pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

[0280] Preparations for oral administration may be suitably formulatedto give controlled release of the active complexes.

[0281] For buccal administration the compositions may take the form oftablets or lozenges formulated in conventional manner.

[0282] For administration by inhalation, the complexes for use accordingto the present invention are conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebuliser, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the complexesand a suitable powder base such as lactose or starch.

[0283] The complexes may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

[0284] The complexes may also be formulated in rectal compositions suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

[0285] In addition to the formulations described previously, thecomplexes may also be formulated as a depot preparation. Such longacting formulations may be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the complexes may be formulated with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

[0286] The compositions may, if desired, be presented in a pack ordispenser device that may contain one or more unit dosage formscontaining the active ingredient. The pack may for example comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.

[0287] Also encompassed is the use of adjuvants in combination with orin admixture with the complexes of the invention. Adjuvants contemplatedinclude but are not limited to mineral salt adjuvants or mineral saltgel adjuvants, particulate adjuvants, microparticulate adjuvants,mucosal adjuvants, and immunostimulatory adjuvants, such as thosedescribed in Section 4.5. Adjuvants can be administered to a subject asa mixture with complexes of the invention, or used in combination withthe complexes as described in Section 4.7.2.

[0288] Also contemplated is the use of adenosine diphosphate (ADP) incombination with or in admixture with the complexes of the invention,preferably gp96 complexes.

[0289] 4.7.4. Kits

[0290] The invention also provides kits for carrying out the methodsand/or therapeutic regimens of the invention.

[0291] In one embodiment, such kits comprise in one or more containersprotein preparations comprising antigenic proteins and peptides forcombining with HSPs and/or α2M that are provided in a second container.In another embodiment, such kits comprise in one or more containersdigested peptides comprising antigenic peptides for combining with HSPsand/or α2M that are provided in a second container. Alternatively,proteins and/or peptides can be supplied in one or more containers forcomplexing to HSPs and/or α2M isolated from a specific patient forautologous administration. Optionally, a purified HSP for complexing toproteins and peptides is further provided in a second container.

[0292] In another embodiment, such kits comprise in one or morecontainers therapeutically or prophylactically effective amounts of thecomplexed proteins/peptides to HSPs and/or α2M, preferably purified, inpharmaceutically acceptable form. The kits optionally further comprisein a second container sensitized APCs, preferably purified.

[0293] The HSP or α2M complexes in a container of a kit of the inventionmay be in the form of a pharmaceutically acceptable solution, e.g., incombination with sterile saline, dextrose solution, or bufferedsolution, or other pharmaceutically acceptable sterile fluid.Alternatively, the HSP and α2M complexes may be lyophilized ordesiccated; in this instance, the kit optionally further comprises in acontainer a pharmaceutically acceptable solution (e.g., saline, dextrosesolution, etc.), preferably sterile, to reconstitute the HSPs and α2M orα2M and HSP-containing complexes to form a solution for injectionpurposes.

[0294] In another embodiment, a kit of the invention further comprises aneedle or syringe, preferably packaged in sterile form, for injectingthe HSP and α2M complex, and/or a packaged alcohol pad. Instructions areoptionally included for administration of α2M and HSP-peptide complexesby a clinician or by the patient.

[0295] Kits are also provided for carrying out the combination therapiesof the present invention. In one embodiment, a kit comprises a firstcontainer containing a purified HSP complexes or α2M prepration and asecond container containing a non-HSP and non-α2M based therapeuticmodality for treatment of cancer. Preferably, the cancer is CML, the HSPcomplexes comprises hsp70-peptide complexes, and the therapeuticmodality is Gleevec™. In a specific embodiment, the second containercontains imatinib mesylate. In another specific embodiment, the imatinibmesylate is purified.

[0296] In a specific embodiment, a kit comprises a first containercontaining a purified HSP complexes or α2M complexes in an amountineffective to treat a disease or disorder when administered alone; anda second container containing a non-HSP and non-α2M based treatmentmodality in an amount that, when administered before, concurrently with,or after the administration of the HSP complexes or α2M complexes in thefirst container, is effective to improve overall treatment effectivenessover the effectiveness of the administration of each component alone. Inanother specific embodiment, a kit comprises a first containercontaining a purified HSP complexes or α2M complexes in an amountineffective to treat a disease or disorder when administered alone; anda second container containing one or more non-HSP and non-α2M basedtreatment modalities in an amount that, when administered before,concurrently with, or after the administration of the HSP complexes orα2M complexes in the first container, is effective to improve overalltreatment effectiveness over the effectiveness of the administration ofthe HSP complexes or α2M complexes administered alone or the treatmentmodalities administered alone. In yet another specific embodiment, afirst container containing a purified HSP complexes or α2M complexes inan amount ineffective to treat a disease or disorder when administeredalone; and a second container and third container, each containing anon-HSP and non-α2M based treatment modality in an amount that, whenadministered before, concurrently with, or after the administration ofthe HSP complexes or α2M complexes in the first container, is effectiveto improve overall treatment effectiveness over the effectiveness of theadministration of HSP complexes or α2M complexes administered alone ortreatment modalities administered alone. In a preferred specificembodiment, the invention provides a kit comprising in a firstcontainer, a purified HSP complexes or α2M comprising a population ofnoncovalent HSP-peptide complexes or α2M-peptide complexes of theinvention; in a second container, a composition comprising ananti-cancer agent; and in a third container, a composition comprising acytokine or an adjuvant.

[0297] The kit may for example comprise metal or plastic foil, such as ablister pack. The kit may be accompanied by one or more reusable ordisposable device(s) for administration (e.g, syringes, needles,dispensing pens) and/or instructions for administration.

[0298] 4.8. Determination of Immunogenicity of the HSP and α2M Complexes

[0299] Optionally, the HSP-protein complexes, HSP-peptide complexes,α2M-protein complexes and α2M-peptide complexes of the invention can beassayed for immunogenicity using any method known in the art. By way ofexample but not limitation, one of the following procedures can be used.In a preferred embodiment, the ELISPOT assay is used (see, infra,Section 4.9.4).

[0300] 4.8.1. The MLTC Assay

[0301] Briefly, mice are injected with an amount of the HSP- and/or α2Mcomplexes, using any convenient route of administration. As a negativecontrol, other mice are injected with, e.g., HSP complexed to proteinsand/or peptides prepared from normal tissue. Cells known to containspecific antigens, e.g. tumor cells or cells infected with an agent ofan infectious disease, may act as a positive control for the assay. Themice are injected twice, 7-10 days apart. Ten days after the lastimmunization, the spleens are removed and the lymphocytes released. Thereleased lymphocytes may be re-stimulated subsequently in vitro by theaddition of dead cells that expressed the antigen of interest.

[0302] For example, 8×10⁶ immune spleen cells may be stimulated with4×10⁴ mitomycin C treated or γ-irradiated (5-10,000 rads) cellscontaining the antigen of interest (or cells transfected with anappropriate gene, as the case may be) in 3 ml RPMI medium containing 10%fetal calf serum. In certain cases 33% secondary mixed lymphocyteculture supernatant may be included in the culture medium as a source ofT cell growth factors (See, Glasebrook, et al., 1980, J. Exp. Med.151:876). To test the primary cytotoxic T cell response afterimmunization, spleen cells may be cultured without stimulation. In someexperiments spleen cells of the immunized mice may also be re-stimulatedwith antigenically distinct cells, to determine the specificity of thecytotoxic T cell response.

[0303] Six days later the cultures are tested for cytotoxicity in a 4hour ⁵¹Cr-release assay (See, Palladino, et al., 1987, Cancer Res.47:5074-5079 and Blachere, at al., 1993, J. Immunotherapy 14:352-356).In this assay, the mixed lymphocyte culture is added to a target cellsuspension to give different effector:target (E:T) ratios (usually 1:1to 40: 1). The target cells are prelabelled by incubating 1×10⁶ targetcells in culture medium containing 20 mCi ⁵¹Cr/ml for one hour at 37° C.The cells are washed three times following labeling. Each assay point(E:T ratio) is performed in triplicate and the appropriate controlsincorporated to measure spontaneous ⁵¹Cr release (no lymphocytes addedto assay) and 100% release (cells lysed with detergent). Afterincubating the cell mixtures for 4 hours, the cells are pelletted bycentrifugation at 200 g for 5 minutes. The amount of ⁵¹Cr released intothe supernatant is measured by a gamma counter. The percent cytotoxicityis measured as cpm in the test sample minus spontaneously released cpmdivided by the total detergent released cpm minus spontaneously releasedcpm.

[0304] In order to block the MHC class I cascade a concentratedhybridoma supernatant derived from K-44 hybridoma cells (an anti-MHCclass I hybridoma) is added to the test samples to a final concentrationof 12.5%.

[0305] 4.8.2. CD4+ T-Cell Proliferation Assay

[0306] Primary T cells are obtained from spleen, fresh blood, or CSF andpurified by centrifugation using FICOLL-PAQUE PLUS (Pharmacia, Upsalla,Sweden) essentially as described by Kruse and Sebald, 1992, EMBO J. 11:3237-3244. The peripheral blood mononuclear cells are incubated for 7-10days with a lysate of cells expressing an antigenic molecule. Antigenpresenting cells may, optionally be added to the culture 24 to 48 hoursprior to the assay, in order to process and present the antigen in thelysate. The cells are then harvested by centrifugation, and washed inRPMI 1640 media (GibcoBRL, Gaithersburg, Md.). 5×10⁴ activated Tcells/well are in RPMI 1640 media containing 10% fetal bovine serum, 10mM HEPES, pH 7.5, 2 mM L-glutamine, 100 units/ml penicillin G, and 100μg/ml streptomycin sulphate in 96 well plates for 72 hrs at 37° C.,pulsed with 1 μCi ³H-thymidine (DuPont NEN, Boston, Mass.)/well for 6hrs, harvested, and radioactivity measured in a TOPCOUNT scintillationcounter (Packard Instrument Co., Meriden, Conn.).

[0307] 4.8.3. Antibody Response Assay

[0308] In a certain embodiment of the invention, the immunogenicity ofan HSP- or α2M-complex is determined by measuring antibodies produced inresponse to the vaccination with the complex. In one mode of theembodiment, microtitre plates (96-well Immuno Plate II, Nunc) are coatedwith 50 μl/well of a 0.75 μg/ml solution of a purified, non-HSP- orα2M-complexed form of the proteins/peptides used in the vaccine in PBSat 4° C. for 16 hours and at 20° C. for 1 hour. The wells are emptiedand blocked with 200 μPBS-T-BSA (PBS containing 0.05% (v/v) TWEEN 20 and1% (w/v) bovine serum albumin) per well at 20° C. for 1 hour, thenwashed 3 times with PBS-T. Fifty μl/well of plasma or CSF from avaccinated animal (such as a model mouse or a human patient) is appliedat 20° C. for 1 hour, and the plates are washed 3 times with PBS-T. Theanti-peptide antibody activity is then measured calorimetrically afterincubating at 20° C. for 1 hour with 50 μl/well of sheep anti-mouse oranti-human immunoglobulin, as appropriate, conjugated with horseradishperoxidase (Amersham) diluted 1:1,500 in PBS-T-BSA and (after 3 furtherPBS-T washes as above) with 50 μl of an o-phenylene diamine (OPD)-H₂O₂substrate solution. The reaction is stopped with 150 μl of 2M H₂SO₄after 5 minutes and absorbance is determined in a Kontron SLT-210photometer (SLT Lab-instr., Zurich, Switzerland) at 492 nm (ref. 620nm).

[0309] 4.8.4. Citokine Detection Assay

[0310] The CD4+ T cell proliferative response to HSP- or α2M-complexesof the invention may be measured by detection and quantitation of thelevels of specific cytokines. In one embodiment, for example,intracellular cytokines may be measured using an IFN-γ detection assayto test for immunogenicity of a complex of the invention. In an exampleof this method, peripheral blood mononuclear cells from a subjecttreated with a HSP-peptide or α2M peptide complex are stimulated withpeptide antigens of a given tumor or with peptide antigens of an agentof infectious disease. Cells are then stained with T cell-specificlabeled antibodies detectable by flow cytometry, for exampleFITC-conjugated anti-CD8 and PerCP-labeled anti-CD4 antibodies. Afterwashing, cells are fixed, permeabilized, and reacted with dye-labeledantibodies reactive with human IFN-γ (PE-anti-IFN-γ). Samples areanalyzed by flow cytometry using standard techniques.

[0311] Alternatively, a filter immunoassay, the enzyme-linked immunospotassay (ELISPOT) assay, may be used to detect specific cytokinessurrounding a T cell. In one embodiment, for example, anitrocellulose-backed microtiter plate is coated with a purifiedcytokine-specific primary antibody, i.e., anti-IFN-γ, and the plate isblocked to avoid background due to nonspecific binding of otherproteins. A sample of mononuclear blood cells, containingcytokine-secreting cells, obtained from a subject treated with aHSP-peptide and/or α2M peptide complex, which sample is diluted onto thewells of the microtitre plate. A labeled, e.g., biotin-labeled,secondary anti-cytokine antibody is added. The antibody cytokine complexcan then be detected, i.e. by enzyme-conjugatedstreptavidin—cytokine-secreting cells will appear as “spots” by visual,microscopic, or electronic detection methods.

[0312] 4.8.5. Tetramer Assay

[0313] In another embodiment, the “tetramer staining” assay (Altman etal., 1996, Science 274: 94-96) may be used to identify antigen-specificT-cells. For example, in one embodiment, an MHC molecule containing aspecific peptide antigen, such as a tumor-specific antigen, ismultimerized to make soluble peptide tetramers and labeled, for example,by complexing to streptavidin. The MHC-peptide antigen complex is thenmixed with a population of T cells obtained from a subject treated witha HSP- or α2M-complex. Biotin is then used to stain T cells whichexpress the antigen of interest, i.e., the tumor-specific antigen.

4.9. Monitoring of Effects During Cancer Prevention and Immunotherapy

[0314] The effect of immunotherapy with HSP- or α2M-complexes on thedevelopment and progression of neoplastic diseases can be monitored byany method known to one skilled in the art, including but not limited tomeasuring: a) delayed hypersensitivity as an assessment of cellularimmunity; b) activity of cytolytic T-lymphocytes in vitro; c) levels oftumor specific antigens, e.g., carcinoembryonic (CEA) antigens; d)changes in the morphology of tumors using techniques such as a computedtomographic (CT) scan; and e) changes in levels of putative biomarkersof risk for a particular cancer in individuals at high risk, and f)changes in the morphology of tumors using a sonogram.

[0315] The following subsections describe optional, exemplaryprocedures.

[0316] 4.9.1. Dielayed Hypersensitivity Skin Test

[0317] Delayed hypersensitivity skin tests are of great value in theoverall immunocompetence and cellular immunity to an antigen. Inabilityto react to a battery of common skin antigens is termed anergy (Sato,T., et al., 1995, Clin. Immunol. Pathol. 74:35-43).

[0318] Proper technique of skin testing requires that the antigens bestored sterile at 4° C., protected from light and reconstituted shortlybefore use. A 25- or 27-gauge needle ensures intradermal, rather thansubcutaneous, administration of antigen. Twenty-four and 48 hours afterintradermal administration of the antigen, the largest dimensions ofboth erythema and induration are measured with a ruler. Hypoactivity toany given antigen or group of antigens is confirmed by testing withhigher concentrations of antigen or, in ambiguous circumstances, by arepeat test with an intermediate test.

[0319] 4.9.2. Activity of Cytolytic T-Lymphocytes In Vitro

[0320] 8×10⁶ Peripheral blood derived T lymphocytes isolated by theFicoll-Hypaque centrifugation gradient technique, are restimulated with4×10⁴ mitomycin C treated tumor cells in 3 ml RPMI medium containing 10%fetal calf serum. In some experiments, 33% secondary mixed lymphocyteculture supernatant or IL-2, is included in the culture medium as asource of T cell growth factors.

[0321] In order to measure the primary response of cytolyticT-lymphocytes after immunization, T cells are cultured without thestimulator tumor cells. In other experiments, T cells are restimulatedwith antigenically distinct cells. After six days, the cultures aretested for cytotoxicity in a 4 hour ⁵¹Cr-release assay. The spontaneous⁵¹Cr-release of the targets should reach a level less than 20%. For theanti-MHC class I blocking activity, a tenfold concentrated supernatantof W6/32 hybridoma is added to the test at a final concentration of12.5% (Heike M., et al., J. Immunotherapy 15:165-174).

[0322] 4.9.3. Levels of Tumor Specific Antigens

[0323] Although it may not be possible to detect unique tumor antigenson all tumors, many tumors display antigens that distinguish them fromnormal cells. The monoclonal antibody reagents have permitted theisolation and biochemical characterization of the antigens and have beeninvaluable diagnostically for distinction of transformed fromnontransformed cells and for definition of the cell lineage oftransformed cells. The best-characterized human tumor-associatedantigens are the oncofetal antigens. These antigens are expressed duringembryogenesis, but are absent or very difficult to detect in normaladult tissue. The prototype antigen is carcinoembryonic antigen (CEA), aglycoprotein found on fetal gut an human colon cancer cells, but not onnormal adult colon cells. Since CEA is shed from colon carcinoma cellsand found in the serum, it was originally thought that the presence ofthis antigen in the serum could be used to screen patients for coloncancer. However, patients with other tumors, such as pancreatic andbreast cancer, also have elevated serum levels of CEA. Therefore,monitoring the fall and rise of CEA levels in cancer patients undergoingtherapy has proven useful for predicting tumor progression and responsesto treatment.

[0324] Several other oncofetal antigens have been useful for diagnosingand monitoring human tumors, e.g., alpha-fetoprotein, an alpha-globulinnormally secreted by fetal liver and yolk sac cells, is found in theserum of patients with liver and germinal cell tumors and can be used asa matter of disease status.

[0325] 4.9.4. Computed Tomographic (CT) Scan

[0326] CT remains the choice of techniques for the accurate staging ofcancers. CT has proved more sensitive and specific than any otherimaging techniques for the detection of metastases.

[0327] 4.9.5. Measurement of Putative Biomarkers

[0328] The levels of a putative biomarker for risk of a specific cancerare measured to monitor the effect of compositions comprising cytosolicand membrane-derived proteins. For example, in individuals at enhancedrisk for prostate cancer, serum prostate-specific antigen (PSA) ismeasured by the procedure described by Brawer, M. K., et al., 1992, J.Urol. 147:841-845, and Catalona, W. J., et al., 1993, JAMA 270:948-958;or in individuals at risk for colorectal cancer CEA is measured asdescribed above in Section 4.5.3; and in individuals at enhanced riskfor breast cancer, 16-α-hydroxylation of estradiol is measured by theprocedure described by Schneider, J. et al., 1982, Proc. Natl. Acad.Sci. ISA 79:3047-3051. The references cited above are incorporated byreference herein in their entirety.

[0329] 4.9.6. Sonogram

[0330] A Sonogram remains an alternative choice of technique for theaccurate staging of cancers.

5. EXAMPLE

[0331] The following experiment demonstrates that complexes of (a)antigenic peptides derived from a cellular fraction, with (b) either HSPor alpha-2-macroglobulin (α2M), are effective at protecting an animalprophylactically from cancer cell growth.

5.1. Materials and Method

[0332] 5.1.1 Protein Purification

[0333] For purification of α2M, serum from mice was diluted 1:1 with0.04M Tris pH 7.6, 0.15M NaCl and applied to a 65 ml Sephacryl S 300R(SIGMA) column equilibrated and eluted with the same buffer.α2M-positive fractions were determined by a dot-blot and the buffer inthe fraction was changed to a 0.01M sodium phosphate buffer pH 7.5 byuse of a PD-10 column. The protein-containing fractions were applied toa Concanavalin A sepharose column. Bound protein was eluted with 0.2Mmethylmannose pyranoside and applied to a DEAE column equilibrated with0.05M sodium acetate buffer. α2M was eluted in a pure form as analyzedby SDS-PAGE and immunoblotting with 0. 13M sodium acetate.

[0334] In some experiments, α2M was purchased from SIGMA.

[0335] Gp96 was obtained by the method described in Section 4.3.3.

[0336] 5.1.2 Tumor Rejection Assays

[0337] All immunizations were done intradermally in 100 μl volume ofPBS. Two immunizations were given one week apart. Seven micrograms ofα2M or 1 μg of gp96 was used per injection either as a complex or alone.Live tumor cells (100,000) were washed free of culture medium,resuspended in PBS and injected intradermally one week after the lastimmunization. Tumors were measured in two dimensions. Half of theaverage was used as the radius of the tumor to calculate the tumorvolume. P values were determined using single-classification analysis ofvariance (ANOVA).

[0338] 5.1.3 Generation of Complexes

[0339] Cell lysate was obtained from live Meth A cells by douncehomogenization followed by ultracentrifugation. 100,000 g supernatantwas treated with 0.1% trifluoroacetic acid (TFA) and 3 mM ATP for 10hours followed by centrifugation in a CENTRICON membrane filter(Millipore) with a 10 kDa cut off limit. Peptides less than 10 kDa(referred to as “MethA10”) were further isolated by binding to a C18reverse phase column, eluting the peptides with methanol, drying thepeptides down in a vacuum, and reconstituting the peptides in a buffersuitable for complexing. Gp96, α2M, or albumin (which was used as acontrol) was heated to 50° C. in the presence of 50 molar excess ofMethA10. The reactions containing the resulting complexes were placed atroom temperature for 30 minutes and then placed on ice. Free,uncomplexed peptide was removed using CENTRICON 50 (Millipore).Complexes thus made were used for immunizations.

5.2. Results

[0340] In this experiment, the Meth A tumor model was used todemonstrate the anti-tumor immunity elicited by gp96-peptide complexes,and α2M-peptide complexes. The antigenic MHC I epitopes of this tumorare unknown. Meth A cell lysates were treated with ATP andtrifluoroacetic acid (TFA) and the fraction of peptides that were lessthan 10 kD (MethA10) was collected and complexed to α2M or gp96 asdescribed above. BALB/c mice were immunized with α2M or gp96,un-complexed or complexed with MethA 10. BALB/c mice were also immunizedwith albumin-MethA 10 or PBS as negative controls. Immunizations weredone twice, one week apart. All mice were challenged intradermally with100,000 live Meth A cells one week after the last immunization. Tumorgrowth was monitored every 5 days up to day 20 after the challenge.TABLE 1 Number of mice Number of mice challenged with Compositions usedin with tumor measurable immunization of mice cells at day 0 tumor atday 20 MethA10 only 5 5 Albumin-MethA10 5 5 PBS 5 5 α2M-MethA10complexes 5 0 Gp96-MethA10 complexes 5 0 Gp96 purified from liver 5 5α2M purified from serum 5 4

[0341] The data in Table 1 shows significant tumor protection in miceimmunized with α2M-MethA10 (p<0.05) or gp96-MethA1 (p<0.05) complexesbut not mice immunized with α2M alone, gp96 alone, albumin-MethA10 orPBS.

5.3. Discussion

[0342] The experiment on immunization against tumors described hereindemonstrates a novel approach to immunotherapy of cancers, whereby anarray of total cellular peptides from the tumor, including self andantigenic peptides, is complexed to an HSP or α2M. Such complexeseffectively stimulated the host's immune system to respond specificallyas shown herein. The data indicate that the utility of this approach inprophylaxis can be extended to treatment of pre-existing disease, aswell as in treatment and prevention of pathogenic infections.

[0343] All references cited herein are incorporated herein by referencein their entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

[0344] Many modifications and variations of this invention can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims along with the full scope ofequivalents to which such claims are entitled.

What is claimed is:
 1. A method of treating or preventing a type ofcancer, comprising administering to a subject in need of such treatmentor prevention a composition comprising a population of complexes, saidcomplexes comprising (a) heat shock protein and/oralpha-2-macroglobulin, and (b) antigenic proteins wherein saidpopulation of complexes were produced by complexing heat shock proteinor alpha-2-macroglobulin to (i) antigenic proteins that are at least 50%of the different proteins present in the cells of said type of cancer,or (ii) at least 50 different proteins present in the cells of said typeof cancer; and administering to said subject at least one treatmentmodality that does not comprise a heat shock protein oralpha-2-macroglobulin.
 2. A method of treating or preventing a type ofcancer, comprising administering to a subject in need of such treatmentor prevention a composition comprising a population of complexes, saidcomplexes comprising (a) heat shock protein and/oralpha-2-macroglobulin, and (b) antigenic peptides wherein saidpopulation of complexes were produced by a method comprising digesting aprotein preparation comprising (i) at least 50% of the differentproteins present in cells of said type of cancer or (ii) at least 50different proteins present in cells of said type of cancer with one ormore proteases to produce a population of antigenic peptides, andcomplexing the population of antigenic peptides to heat shock protein oralpha-2-macroglobulin; and administering to said subject at least onetreatment modality that does not comprise a heat shock protein oralpha-2-macroglobulin.
 3. A method of treating or preventing a type ofcancer, comprising administering to a subject in need of such treatmentor prevention a composition comprising a population of complexes, saidcomplexes comprising (i) heat shock protein and/or alpha-2-macroglobulinand (ii) antigenic peptides wherein said population of complexes wereproduced by a method comprising (a) exposing a protein preparationcomprising (A) at least 50% of the different proteins present in cellsof said type of cancer or (B) at least 50 different proteins present incells of said type of cancer to ATP, guanidium hydrochloride, and/oracidic conditions, to produce a population of antigenic peptides; (b)recovering the population of antigenic peptides; and (c) complexing thepopulation of antigenic peptides to heat shock protein oralpha-2-macroglobulin; and administering to said subject at least onetreatment modality that does not comprise a heat shock protein oralpha-2-macroglobulin.
 4. A method of treating or preventing a type ofinfectious disease, comprising administering to a subject in need ofsuch treatment or prevention a composition comprising a population ofcomplexes, said complexes comprising (a) heat shock protein and/oralpha-2-macroglobulin, and (b) antigenic proteins wherein saidpopulation of complexes were produced by complexing heat shock proteinor alpha-2-macroglobulin to antigenic proteins that are at least 50% ofthe different proteins or at least 50 different proteins present inantigenic cells, a cellular fraction thereof, or viral particles thatexpress an antigenic determinant of an agent that causes the infectiousdisease; and administering to said subject at least one treatmentmodality that does not comprise a heat shock protein oralpha-2-macroglobulin.
 5. A method of treating or preventing a type ofinfectious disease, comprising administering to a subject in need ofsuch treatment or prevention a composition comprising a population ofcomplexes, said complexes comprising (a) heat shock protein and/oralpha-2-macroglobulin, and (b) antigenic peptides wherein saidpopulation of complexes were produced by a method comprising (i)digesting a protein preparation comprising at least 50% of the differentproteins or at least 50 different proteins present in antigenic cells, acellular fraction thereof or viral particles that express an antigenicdeterminant of an agent that causes the infectious disease with either aprotease or a plurality of different proteases; and (ii) complexing thepopulation of antigenic peptides to heat shock protein oralpha-2-macroglobulin; and administering to said subject at least onetreatment modality that does not comprise a heat shock protein oralpha-2-macroglobulin.
 6. A method of treating or preventing a type ofinfectious disease, comprising administering to a subject in need ofsuch treatment or prevention a composition comprising a population ofcomplexes, said complexes comprising (a) heat shock protein and/oralpha-2-macroglobulin, and (b) antigenic peptides wherein said complexeswere produced by a method comprising (i) exposing a protein preparationcomprising at least 50% of the different proteins or at least 50different proteins present in antigenic cells, a cellular fractionthereof, or viral particles that express an antigenic determinant of anagent that causes the infectious disease to ATP, guanidiumhydrochloride, and/or acidic conditions, to produce a population ofantigenic peptides; (ii) recovering the population of antigenicpeptides; and (iii) complexing the population of antigenic peptides toheat shock protein or alpha-2-macroglobulin; and administering to saidsubject at least one treatment modality that does not comprise a heatshock protein or alpha-2-macroglobulin.
 7. The method of claim 1 whereinsaid complexing the population of antigenic proteins to the heat shockproteins is via formation of a covalent bond.
 8. The method of claim 1wherein said complexing the population of antigenic proteins to the heatshock proteins is via formation of a non-covalent bond.
 9. The method ofclaim 2 or 3 wherein said complexing the population of antigenicpeptides to the heat shock proteins is via formation of a covalent bond.10. The method of claim 2 or 3 wherein said complexing the population ofantigenic peptides to the heat shock proteins is via formation of anon-covalent bond.
 11. The method of claim 4 wherein said complexing thepopulation of antigenic proteins to α-2-macroglobulin is via formationof a covalent bond.
 12. The method of claim 4 wherein said complexingthe population of antigenic proteins to α-2-macroglobulin is viaformation of a non-covalent bond.
 13. The method of claim 5 or 6 whereinsaid complexing the population of antigenic peptides to theα-2-macroglobulin is via formation of a covalent bond.
 14. The method ofclaim 5 or 6 wherein said complexing the population of antigenicpeptides to α-2-macroglobulin is via formation of a non-covalent bond.15. The method of claim 1 wherein said population of complexescomprising heat shock protein and/or alpha-2-macroglobulin, andantigenic proteins is purified.
 16. The method of claim 4 wherein saidpopulation of complexes is purified.
 17. The method of claim 2 or 3wherein said population of complexes is purified.
 18. The method ofclaim 5 or 6 wherein said population of complexes is purified.
 19. Themethod of claim 1, 2 or 3, wherein the cells of same type of cancer arefrom a metastasis.
 20. The method of claim 1, 2 or 3, wherein the cancertreated or prevented is a metastasis.
 21. The method of claim 5, 6 or 7,wherein the antigenic cells are infected by the agent that causes theinfectious disease.
 22. The method of claim 5, 6 or 7, wherein theantigenic cells are infected by a variant of said agent, that displaysantigenicity of said agent.
 23. The method of claim 1, 2, or 3 whereinthe at least treatment modality comprises a chemotherapeutic agent, ananti-angiogenic agent, a cytokine, a biological response modifier, ahormone, an antibody, a polynucleotide, an immunostimulatoryoligonucleotide, a photodynamic therapeutic agent or radiation.
 24. Themethod of claim 4, 5, or 6 wherein the at least one treatment modalitycomprises an antibiotic, an antiviral, an antiprotozoal compound, anantifungal compound, an antihelminthic compound, an antibody, acytokine, a hormone, an immunostimulatory oligonucleotide, or apolynucleotide.
 25. The method of claim 1, 2, 3, 4, 5, or 6 wherein saidcomposition is administered before, concurrently with, or afteradministration of the at least one treatment modality.
 26. The method ofclaim 1, 2, 3, 4, 5 or 6 wherein the subject has previously beennon-responsive to treatment with said at least one treatment modality inthe absence of said composition.
 27. The method of claim 1, 2, 3, 4, 5,or 6 wherein said administering of said composition is repeated atweekly intervals.
 28. The method of claim 1, 2, 3, 4, 5, or 6 whereinsaid administering of said composition is repeated at the same site ofthe subject.
 29. The method of claim 1, 2, 3, 4, 5, or 6 wherein saidadministering of said composition is intradermally or subcutaneously.30. The method of claim 1, 2, 3, 4, 5, or 6 wherein a sub-optimal amountof said composition is administered.
 31. The method of claim 1, 2, 3, 4,5, or 6 wherein a sub-optimal amount of said at least one treatmentmodality is administered.
 32. The method of claim 1, 2, 3, 4, 5, or 6wherein the subject is human.
 33. The method of claim 1 wherein theantigenic proteins are autologous to the subject.
 34. The method ofclaim 4 wherein the antigenic proteins are autologous to the subject.35. The method of claim 2 or 3 wherein the antigenic peptides areautologous to the subject.
 36. The method of claim 5 or 6 wherein theantigenic peptides are autologous to the subject.
 37. A kit comprising afirst container containing a composition comprising a population ofcomplexes, said complexes comprising (a) heat shock protein and/oralpha-2-macroglobulin, and (b) antigenic proteins, wherein saidpopulation of complexes were produced by complexing heat shock proteinor alpha-2-macroglobulin to antigenic proteins that are at least 50% ofthe different proteins present in antigenic cells or at least 50different proteins present in antigenic cells; and a second containercontaining a treatment modality that does not comprise heat shockprotein or alpha-2-macroglobulin.
 38. A kit comprising a first containercontaining a composition comprising a population of complexes, saidcomplexes comprising (a) heat shock protein and/oralpha-2-macroglobulin, and (b) antigenic proteins, wherein saidpopulation of complexes were produced by a method comprising (i)digesting a protein preparation comprising at least 50% of the differentproteins or at least 50 different proteins present in antigenic cellswith one or more proteases to produce a population of antigenicpeptides, and (ii) complexing the population of antigenic peptides toheat shock protein or alpha-2-macroglobulin; and a second containercontaining a non-heat shock protein and non-alpha-2-macroglobulin-basedtreatment modality.
 39. A kit comprising a first container containing acomposition comprising a population of complexes, said complexescomprising (a) heat shock protein and/or alpha-2-macroglobulin, and (b)antigenic proteins wherein said population of complexes were produced bya method comprising (i) exposing a protein preparation comprising atleast 50% of the different proteins or at least 50 different proteinspresent in antigenic cells to ATP, guanidium hydrochloride, and/oracidic conditions, to produce a population of antigenic peptides; (ii)recovering the population of antigenic peptides; and (iii) complexingthe population of antigenic peptides to heat shock protein oralpha-2-macroglobulin; and a second container containing a non-heatshock protein and non-alpha-2-macroglobulin-based treatment modality.